Neisseria meningitidis, the leading cause of pyogenic meningitis, can modulate gene expression via uptake of host pro-inflammatory cytokines leading to increased virulence. This uptake is mediated by type IV pili (Tfp) and reliant on the PilT ATPase activity. Two Tfp subunits, PilE and PilQ, are identified as the ligands for TNF-α and IL-8 in a glycan-dependent manner, and their deletion results in decreased virulence and increased survival in a mouse model. We propose a novel mechanism by which pathogens use the twitching motility mode of the Tfp machinery for sensing and importing host elicitors, aligning with the inflamed environment and switching to the virulent state.Interactions between commensal pathogens and hosts are critical for disease development but the underlying mechanisms for switching between the commensal and virulent states are unknown. We show that the human pathogen  An early investigation by Porat et al. [E. coli enhances proliferation of these strains. More recently, Lee et al. [E. coli both in vitro and in vivo, but no underlying mechanism was proposed.ven time . It is tven time ,3. The itime [Nm . Recentl [et al.  demonstrt et al.  demonstre et al.  providedNm expresses virulence factors that allow pathogenic strains to traverse the nasopharyngeal mucosa, disseminate into the bloodstream, and then cross the blood–brain barrier. These virulence factors are broadly grouped into two families: the major adhesin group (which includes Tfp [udes Tfp ) and theudes Tfp ,9.O-linked trisaccharide between amino acid residues 45 and 73 of meningococcal pilin [Nm in contact with host cells increases the transcription of the pptB gene encoding a transferase that adds phosphoglycerol onto Tfp. This post-translational modification is a Tfp-dependent contact between bacteria which use this regulated detachment process to propagate to new colonization sites and migrate across the epithelium during invasion [The meningococcal Tfp is a multi-functional organelle consisting of up to 18 different protein subunits . The Pilal pilin . This stal pilin , but pilal pilin ; howeveral pilin . PilQ, oal pilin  and formal pilin . A phenoal pilin . Prolifeinvasion .Nm is variable. Sialic acid incorporation into lipooligosaccharides (LOS) [An important factor that may potentially alter commensal behaviour towards pathogenicity is the inflammatory state of the host . Moreovees (LOS)  and capses (LOS)  and infles (LOS) ,25. In pNm is highly adaptable to its environment; it downregulates pili expression and capsule production after contact with host cells [Nm in contact with epithelial and endothelial cells has revealed altered expression levels of certain genes involved in pathogenesis, such as IgA1 protease, proteins involved in iron uptake and Tfp assembly [Nm.st cells . Transcrassembly . The cooNm on the host and the inflammatory response caused by Nm infection have been investigated at length; what remains to be considered is how these cytokine-orchestrated immune responses influence Nm and modulate virulence gene expression and phenotype. Here, we investigate the bacterial mechanisms employed to respond to this inflammatory reaction and phenotypic change independently of any cell contact. We show that detection of environmental changes and recognition by Nm ligands, followed by uptake of cytokines, are key steps in the conversion of commensal bacteria into hypervirulent phenotypes.The effects of 3.3.1.Nm, IL-8, IL-10, IL-12, IFN-γ and TNF-α were immobilized in amino-linked enzyme-linked immunosorbant assay (ELISA) plates and incubated with digoxigenin-labelled log-phase-grown Nm strain MC58. The bacterial cells bound to different cytokines with a somewhat variable degree of binding among cytokines (a). TNF-α exhibited the highest binding signal, with the rest of the cytokines exhibiting lower signals with marginal variations. We then examined the concentration-dependent effects of cytokines (using TNF-α and IL-8) on bacterial growth. Bacteria were incubated with TNF-α or IL-8, and the growth was measured for a 24 h period. The mean average results for non-induced and cytokine-induced bacteria, collated from three independent experiments, show that TNF-α or IL-8 had no effect on bacterial growth .To determine the range and specificity of cytokines that bind ytokines a. TNF-α Nm serogroup B MC58 with TNF-α and IL-8. To examine whether this interaction is confined to Nm serogroup B or extends to other serogroups (different types of capsule structure classify Nm into different serogroups), five clinical isolates of serogroups B and Y were selected [b).Our findings reveal an interaction between surface structures of the capsulated selected , and binNm TNF-α-binding adhesin(s); two subunits of the meningococcal Tfp, PilE  and PilQ , were identified as putative human cytokine receptors. To evaluate the contribution of these adhesins to bacterial binding, ΔpilQ and ΔpilE MC58 mutants were generated. The ΔpilQ mutation resulted in a significant reduction in binding to all of the examined cytokines (b). However, the ΔpilE mutation did not result in any significant reduction to cytokine binding with the exception of TNF-α (b). These data suggest that PilQ is an important meningococcal receptor for certain cytokines. However, it was impossible to confirm the important role of PilQ in a complemented strain in ΔpilQ mutant background because of a well-established transformation defect of this mutant. Several attempts using different transformation approaches were unsuccessful in introducing the complementing plasmid within the ΔpilQ mutant strain (data not shown).A retagging technique  was usedytokines b. Howeveof TNF-α b. These 3.2.Nm binding, using TNF-α and IL-8 as examples.Many cytokines possess lectin-like activity that may be essential for the expression of their full biological activities. For example, pili are post-translationally modified  and the surface accessibility of phosphorylcholine on pili is affected by changes to the structure of the pilin-linked glycan . Here, wO-OTase) mutants  of the MC58 strain were generated and examined for binding to IL-8 and TNF-α. All three mutants exhibited significant reductions in binding to both cytokines (c). By contrast, a ΔlgtF mutant of the MC58 strain  [Three oligosaccharyltransferase (ytokines c. By con or LOS)  did not Nm, and to identify the role of glycosylated surface structures of Nm, the inhibition of IL-8- and TNF-α-binding were examined using a series of lectins—Griffonia (Bandeiraea) simplicifolia lectin II (GSL-II); Datura stramonium lectin (DSL); Erythrina cristagalli lectin (ECL); Lycopersicon esculentum (tomato) lectin (LEL); Solanum tuberosum (potato) lectin (STL); Vicia villosa agglutinin (VVA); Artocarpus integrifolia —with different specificities . The ELISA results revealed a significant reduction in Nm binding to TNF-α compared with untreated Nm, with all lectins examined except DSL and LEL (d). In addition, the binding of IL-8 was significantly reduced with ECL and VVA, suggesting that the IL-8 and TNF-α interactions are mediated by different PilE protein glycan moieties (d).To investigate further the nature of the interaction between cytokines and the surface structure of  and LEL d. In addmoieties d.siaD mutant (deficient in capsule formation) was generated, and the adhesion to TNF-α and IL-8 was examined. Binding of both cytokines to MC58 was significantly lower than that to the ΔsiaD uncapsulated isogenic strain (e).To exclude the role of the capsule in binding, a Δc strain e.siaD uncapsulated and O-OTase Nm mutants suggest that the glycosylated structure of Tfp (pilin) plays a critical role in this interaction.Collectively, the results of the lectin inhibition assays and the increased level of binding to cytokines in the ΔE. coli, which possesses different glycosylation machinery than Nm) binds to IL-8 and TNF-α, indicating that it is unlikely to be glycosylated in the same manner as endogenous meningococcal PilQ. These data were consistent with our LC-MS/MS analysis, reinforcing the notion that PilQ is not glycosylated and providing additional evidence for a protein–protein interaction .In addition, purified recombinant PilQ  and 1080 (approx. 45%) Nm genes were altered, respectively ). The genes with modified expression were categorized using the Genome Properties System (http://cmr.jcvi.org/tigr-scripts/CMR/CmrHomePage.cgi). A Log2 ratio of read counts higher than 0.5 was considered to indicate significant differences between induced and non-induced conditions.To investigate whether the bacterial interaction with human cytokines affects 7.1S), energy metabolism (7.2S), transport and binding proteins (7.3S), cell envelope (7.4S), bacterial survival (7.5S), regulatory function (7.6S) and amino acid synthesis (7.7S) were altered. A significant portion of the regulated genes were of unknown function .Among the known genes, the expression of those coding for adhesion  [Meningococcal Serine Protease A (MSPA) [xcel file). Our study also showed that SiaD (NMB0067) and LipA (NMB0082), which are involved in LPS production, are highly upregulated by TNF-α exposure.The comparison of the gene activation profiles in in (APP)  and MeniA (MSPA)   in DMEM in the presence or the absence of cytokines IL-8 or TNF-α (both 100 ng ml−1). The β-galactosidase activity measurements from strains of Nm bearing the promoter NMB0750 , NMB0946  and NMB1998 (mspA)-lacZ fusions confirmed that gene expression of prx and bcp is significantly regulated following the treatment with cytokines (b). The expression of mspA, however, was increased by 22% compared with uninduced bacteria.In batch culture, it is unlikely that the level of expression at the beginning of growth is the same as the steady-state level , becauseytokines b. The ex3.5.Nm. Meningococcus displays a vast repertoire of genes, often involved in virulence, whose expression is controlled by inflammatory cytokines mediated through reversible changes in the Tfp structure. The phenotypes observed here can therefore be due to variation in expression of genes involved in Nm surface structure. A functional study was carried out to validate the deep-sequencing data and differences in the transcriptional profiles observed to assess their role in Nm pathogenicity and/or virulence. To assess whether different cytokine treatments of wild-type (wt) MC58 and its isogenic mutants differ in sensitivity towards bactericidal activity, experiments were performed at the initial log-phase (9 h) of the Nm growth in the presence and the absence of cytokines (c). The rabbit antibody against recombinant PilQ in the presence of rabbit complement was capable of inducing 67% cell death in the untreated wt MC58 strain (i.e. 33% survived), whereas in the case of TNF-α-induced bacteria 31% cell death was induced (i.e. 69% survived). This is equivalent to a doubling of survival. By comparison, in the isogenic  mutants, there was no significant difference between cytokine-treated and non-treated bacteria. IL-8-treated bacteria did not exhibit any significant resistance to complement-mediated lysis (c). These data confirm the significance of modulation of surface structures (hence pathogenicity) and validate the results from deep sequencing.Resistance to complement-mediated lysis is a key virulence attribute of ytokines c. The raed lysis c. These 3.6.Nm, wild-type bacteria and related mutants  were grown in DMEM and induced with either TNF-α or IL-8 (40 ng ml−1) for 9 h at 37°C. Cells were then extensively washed and fixed for transmission electron microscopy. The cytokines were detected with monoclonal antibodies, followed by secondary antibody conjugated with 10 nm colloidal gold particles. The majority of the TNF-α and IL-8-gold labelling was localized in the cytoplasm of wt Nm  had no effect on normal piliation phenotype [No TNF-α or IL-8 uptake was seen in the cytoplasm of the Δhenotype , also coNm wild-type MC58 bacteria was confirmed by confocal imaging, whereas in control experiments there was no uptake of labelled Gal-3 , confirming the specificity of the uptake (a). Furthermore, pre-treatment of the bacteria with non-labelled TNF-α inhibited the uptake of labelled TNF-α (a), further confirming uptake specificity.The intracellular uptake of labelled TNF-α by e uptake a. Furtheed TNF-α a, furtheNm isolated directly from the peritoneum and blood of mice 4 h post-infection exhibited TNF-α uptake, whereas ΔpglC/L mutant Nm did not in EM experiments (b). Immunogold staining demonstrated 66% and 50% of the wt MC58 bacterial population in peritoneum and blood, respectively, showing uptake of TNF-α, but none of the examined samples from the ΔpglC/L mutant showed any signs of TNF-α binding and/or uptake. Collectively, our data confirmed the specific uptake of TNF-α in vivo.In addition, MC58 eriments b. Immuno3.7.Nm genome, acting as transcription modulators for the expression of virulence genes such as autotransporter proteins MSPA and APP, in addition to the PptB transferase that adds phosphoglycerol onto Tfp [We hypothesized that human cytokines may bind across the onto Tfp . Qualitaonto Tfp  was usedpilE, ΔpilQ and ΔpglC/L mutants were grown in the presence or the absence of TNF-α, and antibodies directed against TNF-α were used to select protein cross-linked DNA fragments that were further purified using magnetic beads coated with a secondary antibody. Q-PCR was carried out using primers specific for the app, mspA and pptB promoter regions. The app, pptB and mspA immunoprecipitated DNA fragments from the TNF-α-induced wt strain were more than four- to fivefold enriched compared with the equivalent non-induced samples (a). These data confirm the uptake of TNF-α by wt Nm and suggest that TNF-α binds to genomic DNA sites within the app, mspA and pptB genes.The wt MC58 strain and the Δ samples a. These app gene. Specific binding was detected with two fragments from the intergenic region immediately upstream from the app gene, but not with the other two fragments (b–e). The alignment of the two binding fragments suggests several conserved binding sites, which require further study to exquisitely define the precise sequence of the DNA-binding site (f).A gel-shift assay was used to study the direct binding of TNF-α to three partially overlapping fragments (70–84 bp) from the intergenic (promoter-containing) and one from the intragenic (non-promoter containing) regions of the ragments b–e. The ing site f.3.8.Nm interact with CD46, a human cell–surface protein involved in the regulation of complement activation. CD46, with the cytoplasmic tail Cyt-2, regulates T-cell-mediated inflammatory responses [Neisseria [The pili of esponses , is prefeisseria . The inteisseria . Transgeeisseria .Nm surface structures required for binding to human cytokines on bacteraemia and meningitis progression was investigated in CD46 transgenic mice. Eight mice in each group were intraperitoneally infected with wt or one of the isogenic mutants  and examined for 72 h. All of the mice developed bacteraemia , but lethal instances of disease occurred only in mice that were infected with MC58 or the ΔpilE mutant. Mice that were infected with ΔpilQ and ΔpglC/L mutants exhibited significantly less mortality and enhanced survival rates (a). These results further confirmed the importance of PilQ and surface glycans for Nm virulence.The impact of the al rates a. These pilQ and ΔpglC/L Nm mutants can still activate immune cells to secrete inflammatory cytokines just like the wt strain. Collectively, our data suggest that although the Nm mutants can still activate cytokine secretion they are at the same time less responsive to cytokines (because they are lacking the specific adhesins); therefore, animal survival is enhanced.In addition, blood samples were collected at 2, 6, 24 and 48 h post-infection to analyse the production of inflammatory cytokines such as TNF-α, IFN-γ, IL-6, keratinocyte chemoattractant  and anti-inflammatory cytokine IL-10 . The results showed that the Δapp, mspA and pptB using Q-PCR. The results clearly showed that mice infected with wt MC58 were expressing these genes significantly higher than the isogenic mutants (b).Furthermore, the total RNA was purified from all collected blood (6 h post-infection) from each group of mice and pooled in one sample, in order to examine the expression of virulence genes such as  mutants b.4.Nm is usually a commensal bacterium of the nasopharynx. During the commensal state, most of the dividing bacteria belong to the same antigenic type and express low levels of virulence genes. Most probably, a peak of pathogenic status (bacteraemia followed by meningitis) is reached when the bacteria sense danger caused by a hyperinflamed environment. Here, we propose that host elicitors in response to microbes are engaged by commensal Nm and control the patterns of pathogenesis. Progression towards pathogenic states requires monitoring of long-term changes of bacterial behaviour to understand how this cycle varies across a broad inflammatory status. Our data provide novel insights into the changes taking place as inflammation continues to rise, and the extent and duration of inflammatory markers change.TNF-α and IL-8 are pro-inflammatory cytokines that have numerous biological activities  and playNm probably employs two mechanisms for binding to IL-8 and TNF-α. Cytokines contain lectin-like carbohydrate domains which are spatially distinct from cytokine-receptor binding sites [Nm might use this strategy to facilitate the neutralization and/or inactivation of human cytokines and the modulation of its own gene expression in response to environmental alterations.ng sites . The lecng sites –46. Nm mpglL, ΔpglC and a ΔpglC/L double mutant), indeed suggest that Nm-cytokine binding is mediated partially by glycan moieties and by protein–protein interactions. This observation is consistent with the findings of Estabrook et al., who showed that mannose-binding lectin binds to the non-glycosylated Nm outer-membrane proteins Opa and PorB in a carbohydrate-independent manner [Nm is mediated by pilus assembly proteins (i.e. PilQ and PilE proteins) and that the virulence properties of Nm are enhanced as a consequence of TNF-α or IL-8 binding and uptake. The ingested cytokines directly bind to genomic DNA, and consequently regulate the expression of several genes.Our data, with a series of glycosyl-transferase-deficient mutants . This is consistent with a previous report showing that IL-8 binding to neutrophil receptors can be prevented by anionic polymers, such as DNA and actin [TNF-α was shown to exhibit DNA-binding activity d,e. Thisnd actin . The avaFurthermore, we show that TNF-α is able to regulate bacterial gene expression. There is a variety of mechanisms that bacteria use to modulate gene expression in response to environmental stimuli. DNA binding by cytokines may change promoter architecture, which could then change the preference (negatively or positively) of RNA polymerase for certain promoters, physically prevent direct binding of RNA polymerase to promoters or prevent binding of transcription factors (gene enhancers or silencers). Indeed, many combinations of these mechanisms may work for different promoters of different virulent (and other) genes to affect global transcription. In addition, in bacteria there are several non-specific DNA-binding proteins, known as nucleoid-associated proteins (NAPs), that bind globally to the bacterial genome (nucleoid), and affect both its structure and its transcriptional activity at the global level . This was consistent with EM analysis of isolated bacteria from the peritoneum and blood samples of post-infected mice. Four hours post-infection inflammation is increased in the peritoneum and 66% of wt MC58 show TNF-α uptake compared with no uptake in the ΔpglC/L mutant (b). This is significant considering that in vivo many different cytokines could potentially interact with Nm in a complex manner, causing a wide range of changes in bacterial behaviour. Despite this complexity, we still see a considerable number of bacteria specifically interacting with TNF-α in the wt MC58 but not in the ΔpglC/L mutant isolated from infected animals.The fact that mice infected with the ΔL mutant b. This iHowever, no significant difference in bacterial levels and cytokine levels in the blood from infected mice were observed between the examined strains , showing that survival of the animals is not related to bacteraemia.pilE mutant, no gold particle uptake was observed . However, the negative staining indicated that there was another form of pili-like surface appendages in the ΔpilE mutant, which could potentially be an alternative entry point for cytokines in vivo, which could affect survival (illustrated in figure 7a).In the case of the ΔPseudomonas aeruginosa, Helicobacter pylori and entero-pathogenic E. coli to a broad range of human cytokines was studied in parallel and the corresponding adhesins were identified . However, the detailed mechanisms of how these opportunistic pathogens actively sense alterations in the host and respond by enhancing their virulence phenotype remain to be studied.The Tfp system is widely distributed across many different bacteria species (more than 150 species), which suggests that cytokine entry into bacteria may be of wide applicability across many species. The binding of b). Our findings also provide a mechanism to explain the frequent development of meningitis in patients with an intense and protracted inflammatory response. Further research comparing the nature of hypervirulent lineages may elucidate the extent to which this feature contributes to the epidemiological distinctiveness of meningococcal infections.Taken together, the proposed mechanism potentially has general applicability and the fundamental knowledge gained from investigations of the uptake system of this bacterium with biochemical analyses should yield substantial insights that will help to unlock more of the secrets of the lifestyle and pathogenic potential of this organism and, potentially, other pathogenic bacteria  .−1)  were coupled to 96-well plates by adding 100 µl of cytokine solution (3 µg ml−1) diluted in sodium carbonate buffer (pH = 9) to each well. The bacterial strains MC58, ΔpilQ, and ΔpilE, ΔpglC/L were labelled with 10 µg ml−1 digoxigenin in PBS for 2 h at room temperature. A total of 100 µl of labelled bacteria (OD600 0.02) were added to each well, and the plates were incubated overnight at 4°C.Purified, human recombinant IL-10, IL-12, IL-8 (aa 1–77), TNF-α (aa 77–233),  and INF-γ  was added to each well. The absorbance at 405nm was measured at different time points. For inhibition assay, the bacterial cells was pre-incubated with a series of lectins  for 2 h followed by several washes, then the bacterial cells were added to coated plates with various cytokines. The following steps were similar to ELISA.The plate was incubated with a polyclonal anti-digoxigenin  in 1% BSA/PBS. Then, 100 µl of ABTS solution  Nm were treated with either TNF-α or IL-8 for 4–9 h, with untreated replicates included for each strain/treatment combination. Bacteria were then fixed in 3% paraformaldehyde and 0.1% glutaraldehyde in phosphate buffer for 2 h at room temperature and processed for transmission electron microscopy. The samples were subsequently washed, dehydrated and processed into Araldite resin blocks (TAAB Laboratories), before being sectioned and mounted onto nickel grids. For the immunogold labelling, samples were washed (in a 1% BSA and 5% goat serum solution) and incubated overnight at 4°C with either anti-TNF-α or anti-IL-8 monoclonal antibodies at 2 µg ml−1 (Thermo Scientific), followed by labelling with goat anti-mouse IgG : 10 nm gold  at 0.2 µg ml−1 for 4 h at 4°C. To prevent non-specific binding with both primary and secondary antibodies, BSA and goat serum were used at appropriate stages in the procedure. Imaging was performed on an FEI Technai 12 Biotwin transmission electron microscope at 100 kV.Wild-type and mutants  and its promoter region were isolated by PCR. Overlapping fragments used here are: Pro.1–2: TTT CGG TTG TCC GTT TGT CGG TTG TTT TCA TTA TTT TTC CTT ATC TGA CGG GAT TCG GGT TTG TTT GGG AT, Pro. 3–4: CGG GAT TCG GGT TTG TTT GGG AGG GCG CGG CTT CCG CTT CCG GGC GGC GCG CGG GAT GTG CCT ATA TGT GCG GTT CGG CG, Pro. 5–6: TAT GTG CGG TTC GGC GTT CGG GCG GAT ATG AAG CAC GCC CTA GGA TTT GTC ATT AAT TTT TGC CTT GGT CTC GGC TTC TTC CA, Internal region of app gene itself: GTT TTT TAC CAA CAG GAG GCT CAT TTG GCG ACA GTG GCT CAC CAA TGT TTA TCT ATG ATG CCC AAA AGC AAA AGT G. These were amplified and radioactively labelled using 32P-γATP and T4 polynucleotide kinase (New England Biolabs). Binding reactions were carried out with 50 000 cpm (approx. 0.1 ng) of 32P-end-labelled oligonucleotide for 20–30 min at room temperature in 10 or 20 μl reaction volumes containing 12% glycerol, 12 mM HEPES-NaOH (pH 7.9), 60 mM KCl, 5 mM MgCl2, 4 mM Tris–Cl (pH 7.9), 0.6 mM EDTA (pH 7.9) and 0.6 mM dithiothreitol.Electrophoretic mobility shift assays (EMSAs) were performed essentially as described . BrieflyProtein–DNA complexes were resolved in 6% polyacrylamide gels pre-electrophoresed for 30 min at room temperature in 0.25 × TBE buffer . Gels were dried and visualized using a phosphorimager.5.5.Total RNA was enriched via two rounds of ribodepletion using a MICROBExpress kit (Ambion). Barcoded RNA-seq libraries were then constructed from each enriched RNA sample using a Total RNA-seq kit (Ambion) and sequenced using a SOLiD 4 genome analyser (Applied Biosystems).Nm MC58) using BioScope v. 1.3.1 software. The htseq-count script from the HTSeq Python package (http://www-huber.embl.de/users/anders/HTSeq/doc/count.html) was used to count the number of reads that were mapped to each gene. The total number of reads per kilobase per million mapped reads were also calculated [The SOLiD reads were mapped to the reference genome , the promoter was amplified with primers 5′-AAAAGGATCCAGCACCCAAATCCACA-3′ and 5′ AAAAGGATCCCCGGTACGATCTTGCAAA-3′. For gene NMB0750 , the promoter was amplified with primers 5′ AAAAGGATCCACATCCATAGTCCTAC-3’ and 5’ AAAAGGATCCTGTGAGGAGAGAATC-3′.Promoter–n pLAS94 . For genmspA), the promoter was amplified with 5′-CGCGGATCCCGCATGATGATTATCCGTGTA-3′ and 5’-CGCGGATCCAAC AACCGGAAAACGCAG-3′ by these sets of primers. Derivatives of pLES94 containing the promoter region were checked by sequencing and transformed into Nm MC58, and verified by PCR. β-galactosidase activity was assayed by the method of Miller [Nm strains for 0–12 h in DMEM medium in the presence or the absence of cytokines IL-8 or TNF-α, both at a concentration of 100 ng ml−1.For gene NMB1998  can develop meningococcal disease [Nm MC58 and the mutant strains were suspended in GC liquid, and each mouse was challenged intraperitoneally (i.p.) with 1.2×109 CFU in 100 μl of GC liquid medium. Experiments were performed with 6–8-week-old mice (n = 8 mice per group). In the control group, mice were challenged i.p. with 100 μl GC liquid. The health condition of all mice was closely monitored for 7 days. At the indicated time points, whole blood samples were collected from the tail vein for the measurement of cytokines, chemokines and bacteraemial levels.Previous studies have shown that the hCD46Ge transgenic mouse line . Whole blood anticoagulated with heparan sulfate was collected by retro-orbital bleeding at 4 h post-infection. Blood cells were removed by gentle centrifugation; bacteria in plasma were washed once with PBS following fixation with 3% PFA, 0.1% glutaraldehyde in phosphate buffer.Bacterial strains MC58 and Δ


From all subjects, saliva level of the tested biomarkers was determined by ELISA. Salivary profiles were assessed in ELP patients by ELISA after being treated with prednisone. A significantly higher level of IFN-γ (P ≤ .01), TNF-α (P ≤ .0001), and sTNFR-2 (P ≤ .01) was detected in ELP patients before treatment than in controls. Following treatment, the salivary levels of IFN-γ (P ≤ .01), TNF-α (P ≤ .05), and sTNFR-2 (P ≤ .01) decreased significantly when compared to their pretreatment levels. This study demonstrated that salivary IFN-γ, TNF-α, and sTNFR-2 can be detectable in ELP patients and decreased significantly after treatment with prednisone, which may reveal the possibility of using these disease-related biomarkers in diagnosis and monitoring.To explore the feasibility of detecting salivary levels of IFN- Althougs in OLP . Evidencs of OLP , 4. Beinronicity , 5. α is a highly pleiotropic, multifunctional cytokine that regulates diverse cellular responses via binding to two distinct cell surface receptors: TNF receptor-1 (TNFR-1/60 kDa) and TNF receptor-2 (TNFR-2/80 kDa) [α-mediated cytotoxic phenomenon occurring through the TNFR-1 signaling pathway [+ cytotoxic T-cells to trigger keratinocyte apoptosis in OLP included the T-cell-secreted TNF-α binding to TNFR-1 on the keratinocyte surface. This binding may activate the keratinocyte caspase cascade resulting in keratinocyte apoptosis. Cell-mediated immunity in OLP may be regulated by various cytokines and their receptors . TNF-α i1 TNFR-1/0 kDa and pathway . Sugermaα-mediated apoptosis [Previous studies showed that many cell types coexpress TNFR-1 and TNFR-2 and require collaboration between these two receptors to generate TNF-poptosis , 9. Furtpoptosis , 11. Accpoptosis .Saliva has more potential diagnostic value that is generally appreciated and has promise to be an aid in the diagnosis of systemic and oral diseases in the last decade . Whole sα, IFN-γ, and sTNFR-2 in saliva of erosive OLP (ELP) patients and to monitor the therapeutic response by determining their levels before and after treatment with a systemic corticosteroid, prednisone.It is important to evaluate the practical application of salivary biomarkers' analyses in clinical management of OLP . AccordiThe entire study sample comprised 40 subjects: 20 patients suffering from ELP  and 20 healthy controls . The patients were diagnosed with ELP; while the controls were healthy normal individuals free form any systemic disease or inflammatory oral lesions. The distribution of subjects according to the age and gender is presented in Subjects were selected from the Outpatient Clinic, Department of Oral Medicine and Periodontology, Faculty of Oral and Dental Medicine, Cairo University. A detailed medical history of each subject was obtained according to the detailed questionnaire of the modified Cornell Medical Index . All subAll patients had oral symptoms, and every case was clinically diagnosed as ELP. Duration of the disease ranged from 2 to 3 months with periods of remission and exacerbation. The erosive lesions were bilateral and extended to involve the buccal mucosa, labial mucosa, and tongue which varied from one patient to the other. Diagnosis was confirmed by histopathologic examination according to the World Health Organization's (WHO's) clinicopathological diagnostic criteria for LP . According to Carbone et al. , ELP patμm low protein binding membrane, separated into 0.5 mL aliquots and frozen at −80°C until assayed.Collection of whole unstimulated saliva (WUS) was done using standard techniques according to Navazesh . At the γ, TNF-α, and sTNFR-2 in saliva of patients with ELP before and after treatment with prednisone. After collecting all the salivary samples, the studied biomarkers were assessed by ELISA.The evaluation in this study included assessing the levels of IFN-γ has been precoated onto a microplate. Standards and samples (100 μL each) were pipetted into the wells in duplicate, and any IFN-γ present was bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for IFN-γ (200 μL) was added to the wells for 2 hours. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution (200 μL) was added to the wells for 30 min and color developed in proportion to the amount of IFN-γ bound in the initial step. The yellow color development was stopped, and the intensity of the blue color was measured by measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm. The minimum detectable dose (MDD) of IFN-γ was typically less than 8.0 pg/mL. The concentrations of samples were calculated from the standard curve, and the results were presented in picogram per milliliter (pg/mL). The concentrations of the curve were ranged from 0–1000 pg/mL.Saliva samples were determined by using a commercially available enzyme-linked immunosorbent assay (ELISA) kit . This assay employs the quantitative sandwich enzyme immunoassay technique. A polyclonal antibody specific for IFN-α and sTNFR-2 in saliva samples were determined by using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems Inc.). These are standard “sandwich” ELISAs and were performed using human recombinant standards according to the manufacturer's instructions. These assays employ the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for TNF-α and sTNFR-2 has been precoated onto a microplate. Standards and samples (100 μL each) in duplicate, were pipetted into the specific wells, and any TNF-α and/or sTNFR-2 present was bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody (200 μL) specific for TNF-α and sTNFR-2 was added to the specific wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution (200 μL) was added to the specific wells and yellow color developed in proportion to the amount of TNF-α and/or sTNFR-2 bound in the initial step. The color development was stopped, and the intensity of the blue color was measured. These assays have a lower limit of detection for TNF-α (1.6 pg/mL) and for sTNFR-2 (0.6 pg/mL). Concentrations of the cytokine and its soluble receptor in the sample were determined by generation of a standard curve for comparison, and results were presented in pg/mL. The TNF-α standard curve ranged from (0–1000 pg/mL) while that of sTNFR-2 ranged from (0–500 pg/mL)Levels of TNF-Results were expressed as mean ± standard deviation (SD) and median. First “one way ANOVA” was performed to demonstrate the difference of each biomarker level between ELP and control group in saliva. Then, “repeated measures ANOVA” was used for comparison of the change of each biomarker level in saliva before and after treatment in ELP group. All data were processed with a computerized statistical package  .γ, TNF-α, and sTNFR-2 were detected in all saliva samples obtained from healthy subjects. The levels of the IFN-γ, TNF-α, and sTNFR-2 (P = .009) in saliva increased remarkably in the ELP group than those in the controls. The highest significance (P = .0001) was observed when both TNF-α and IFN-γ concentrations in saliva were compared to their corresponding control groups (γ (P = .001), TNF-α (P = .0263), and sTNFR-2 (P = .01) decreased significantly when compared to their pretreatment levels (IFN-l groups . Followi P = .000 was obse P = .026, and sTNγ, TNF-α, and sTNFR-2 in ELP patients in response to treatment with prednisone in an attempt to reveal the possibility of their use in disease diagnosis and monitoring. Since systemic corticosteroids are indicated for patients with severe erosive or atrophic OLP [Salivary assays have shown to have a significant correlation with lesional tissue partners  and seruphic OLP  and widephic OLP  owing tophic OLP , 27, prephic OLP . α and INF-γ in OLP samples compared to normal control. These data were consistent with their enhanced expression previously reported from different media in OLP patients which supports their important role in the pathogenesis of OLP [α in OLP patients [α in OLP patients compared to healthy controls which even showed correlation with disease severity being significantly higher in the erosive/atrophic type than in reticular type of OLP. The NF-κB-dependent cytokines TNF-α, IL-1, IL-6, and IL-8 were also detected at significant elevated levels in three types of oral fluids, including saliva, from OLP patients [The present study demonstrated a statistically significant elevation in salivary levels of TNF-s of OLP , 28, 29.patients , 16, 18.patients  demonstrpatients  even sugγ in OLP have been inconsistent. The data presented in this study was supported by a recent investigation showing that erythematous/ulcerated OLP patients had a significant higher level of IFN-γ in lesions and saliva than control [γ in saliva of patients with OLP claiming that it may result from the counter-regulation of pathologically elevated Th2-derived cytokines such as IL-4, involved in cellular immunosuppression. In contrast, Khan et al. [To date, the results on IFN- control . On the  control  recentlyn et al.  could noα 10- to 20-fold greater than those in keratinocytes from normal gingiva [The increase of salivary cytokines in ELP patients observed in this study might be due to local production from cells of the inflammatory infiltration and/or by the keratinocytes themselves . It has  gingiva . α and IFN-γ after treatment with prednisone which advocates the fact that they play an important role in the inflammatory process and immunopathogenesis of OLP. Accordingly, it has been reported that the number of TNF-α-positive mononuclear cells in erosive or atrophic OLP patients significantly decreased after treatment with 0.1% fluocinolone acetonide [γ was significantly elevated in OLP patients and decreased after treatment with 0.1% fluocinolone acetonide. Our results are also consistent with Rhodus et al. [κB-dependent cytokines in patients with ELP following treatment with dexamethasone mouth wash. The authors indicated that detection of disease-related cytokines in saliva indeed has some clinical potential in monitoring therapeutic response and disease activity status of OLP, which is strongly supported by the data presented in this study.The current study illustrated a significant decrease in the salivary levels of TNF-cetonide . Recentlcetonide  also dems et al.  who founα has a role in carcinogenesis and that ELP has a greater rate of malignant evolution, this stresses on the diagnostic and prognostic potential of salivary TNF-α and its receptors in OLP patients [Considering that TNF-patients , 33. Ourα may trigger apoptosis in OLP through inducing differentiation and activation of CD8+ cytotoxic T-cells [α actively drives lymphoproliferation by signaling through TNFR-2. This conclusion is supported by earlier studies suggesting that TNFR-2 transmits signals important for the proliferation of cytotoxic T-cells and excluded an absolute requirement of a coexpression of TNFR-1 for TNFR-2 signaling [α-induced apoptosis requires TNFR-1 signaling [α-induced apoptosis. Several mechanisms have been proposed to explain this observation, including TNFR-2 serving as high affinity trap for TNF-α that delivers TNF-α to TNFR-1 “ligand-passing” [α [TNF- T-cells . Smyth a T-cells  offered ignaling , 36. Whiignaling –40. It hpassing” , TNFR-2 sing” [α , 42, andsing” [α . α might trigger keratinocyte apoptosis in OLP via both TNFR-1 and TNFR-2. The present study, in which the authors duly recognize that the sample size was relatively small, presents some preliminary results related to the significant expression of salivary sTNFR-2 in ELP patients which may give some new insights to the pathogenesis of OLP. Although further conclusions from these data must be interpreted cautiously, additional studies are recommended for evaluating the TNFR-1/TNFR-2 ratio and better understanding of the role of sTNFR-2 in the pathogenesis of OLP.In light of the above-mentioned data, this study suggests that TNF-γ, TNF-α, and sTNFR-2 can be detectable in ELP patients and decreased significantly after treatment with prednisone. The results of this study from saliva of ELP patients provide a potential for specimens to be obtained easily and noninvasively, as well as disease-related biomarkers which may reveal the possibility of their use in diagnosis and monitoring.The current study demonstrated that the absolute concentrations of salivary IFN-The authors report no conflict of interests related to this study.


Tumor necrosis factor α (TNF-α) promotes bone loss and inhibits bone formation. Osterix  is a transcription factor required for osteoblast (OB) differentiation because deletion results in a cartilaginous skeleton. We previously described a TNF suppressor element in the Osx promoter that was used to isolate nuclear proteins mediating TNF inhibition of OB differentiation. Nuclear extracts from TNF-treated pre-OBs were incubated with the TNF suppressor element for protein pull-down, and tryptic fragments were analyzed by mass spectrometry. Chromatin immunoprecipitation (ChIP) assay confirmed eight bound transcription factors. One protein, the paired related homeobox protein (Prx1), had been shown previously to have a critical role in limb bud formation and skeletal patterning. PCR revealed Prx1 expression in primary stromal cells (MSCs), C3H10T1/2 cells, and MC3T3 preosteoblasts. TNF stimulated a 14-fold increase in mRNA for Prx1, rapid cell accumulation in MC3T3 cells, and expression in periosteal and trabecular lining cells in vivo. Transient expression of Prx inhibited transcription of Osx and RUNX2. Expression of the Prx1b isoform or Prx2 decreased Osx and RUNX2 mRNA and OB differentiation in preosteoblasts. Silencing of Prx1 with siRNA abrogated TNF suppression of Osx mRNA and increased basal Osx expression. Electrophoretic mobility shift revealed Prx1b as the preferred isoform binding the Osx promoter. These results identify the homeobox protein Prx1 as an obligate mediator of TNF inhibition of Osx and differentiation of OB progenitors. Activation of Prx1 by TNF may contribute to reduced bone formation in inflammatory arthritis, menopause, and aging. © 2011 American Society for Bone and Mineral Research. Osteoblasts are derived from pluripotent precursors of mesenchymal lineage. In adult bone, fracture healing and renewal recapitulate the developmental steps of bone formation, including recruitment and differentiation of new bone-forming osteoblasts (OBs). OBs firsRUNX2 transcription by 50% and also stimulates a smurf1-mediated ubiquitination/degradation of RUNX2, leading to a 90% reduction in RUNX2 protein in vitro and in vivo. has an important role as a mediator of skeletal damage in inflammatory arthritis.,21,22 TN in vivo. TNF-α inby TNF-α.,16,18 ThWe recently evaluated the structure of the Osx promoter to identify the mechanism of TNF-α inhibition. Since re2+ and Mg2+), and the Amaxa Nucleofector device and reagents, Lonza . siRNA to Prx1, Prx2, and negative-control siRNA were from Ambion/Applied Biosystems , and siRNA transfection reagent was from Santa Cruz Biotechnology, Inc. . Superfect transfection reagent, RNeasy Micro Kit, and Taq PCR Core Kit were from Qiagen . Oligonucleotides and primers were purchased from Eurofins MWG Operon . Real-time PCR was performed on the Bio-Rad MyiQ using the iScript cDNA Synthesis Kit and iQ SYBR Green Supermix . Other reagents were obtained from Sigma-Aldrich . The −1269/+91 Osx promoter-luciferase reporter (Osx–Luc) and deletion mutants were described previously.(MC3T3-E1 (clone 14) mouse preosteoblast cells were obtained from Dr Renny Franceschi , and C3H10T1/2 cells were obtained from ATCC . The sources of reagents were as follows: human TNF-α, PeproTech ; minimal essential medium (MEM) and α-MEM, Gibco/Invitrogen ; heat-inactivated fetal bovine serum (FBS), HyClone ; and trypsin/versene, phosphate-buffered saline  and resuspended in 0.1% trifluoroacetic acid (TFA) prior to mass spectrographic (MS) analysis.The oligo precipitation protocol was performed as described previously with minor modifications. C3H10T1/Tandem mass spectrometry and bioinformatic analyses–liquid chromatography (LC) were performed using a Dionex UltiMate 3000 LC system  coupled online with a linear-ion-trap (LIT) mass spectrometer (MS) . Separations of each digest were performed on a 75-µm-inner-diameter × 360-µm-outer-diameter × 10-cm-long fused silica capillary column , with 5-µm, 300-Å pore-size Jupiter C-18 stationary phase  with an integrated electrospray ionization (ESI) emitter tip. After injecting 5 µL of sample, the column was washed with 98% mobile phase A (0.1% formic acid in water) for 20 minutes, and peptides were eluted by development of a linear gradient of 2% mobile phase B (0.1% formic acid in acetonitrile) to 42% mobile phase B in 140 minutes and then to 98% mobile phase B in an additional 10 minutes, all at a constant flow rate of 250 nL/minute. The column was washed for 20 minutes with 98% mobile phase B and equilibrated with 98% mobile phase A prior to subsequent sample loading.m/z 350 to 1800) was followed by seven MS/MS scans, where the seven most abundant peptide molecular ions were selected for tandem MS using a relative collision-induced dissociation (CID) energy of 35%. Dynamic exclusion was used to minimize redundant selection of peptides for CID.The MS was operated in a data-dependent MS/MS mode in which each full MS scan (precursor ion-selection scan range of Mus musculus proteome database (www.expasy.org) using SEQUEST . Peptides were considered legitimately identified if they achieved a specific charge state and proteolytic cleavage–dependent cross-correlation (Xcorr) scores of 1.9 for [M + H]+1, 2.2 for [M + 2H]+2, and 3.1 for [M + 3H]+3 and a minimum delta correlation score (ΔCn) of 0.08. These stringent filters resulted in a false discovery rate of less than 1%, as determined by searching a reversed human proteome database, as described previously. in MEM and 10% FBS, as described previously.(MC3T3-E1 clone 14 (MC3T3) or C3H10T1/2 cells were plated at 5 × 10eviously. TNF-α waeviously.RNA was prepared using the RNeasy Micro Kit on the days indicated for each experiment. Osx and RUNX2 promoter-luciferase reporters were described previously.,19 QuantRenilla luciferase data to correct for transfection efficiency. For transfection of expression vectors, the same technique was used with exclusion of Renilla luciferase vector. Addition of siRNA or randomized control was done 48 hours prior to treatment with TNF-α using siRNA transfection reagent. The medium was changed every 48 to 72 hours.For reporter assays, cells were transfected with a mixture of Superfect transfection reagent, medium, promoter reporter, and pRL-TK control vector. Cells were harvested 48 hours after transfection and assayed using the Dual Luciferase Assay System. Firefly luciferase values were normalized to In vitro transcription/translation was done using the TNT T7 Coupled Reticulocyte Lysate System according to the manufacturer's instructions.Electrophoretic mobility shifts (EMSAs) were done as described previously.,49 Probe6 C3H10T1/2 cells for each pull-down reaction. Detection of the antibody pull-down DNA complex of interest was done by PCR using 10 µL of DNA per sample and the Taq PCR Core Kit at 40 cycles of 95°C, 30 seconds; 60°C, 30 seconds; and 72°C, 1 minute. Primers spanned the TNF-α response region of the Osx gene and upstream or downstream control sequences. Primer sequences were TNF-α response region (forward) 5′-GACTC AGAAG GAATG ATAGA GGCT-3′ and (reverse) 5′-AGTTA CAATC TAGAG GCAGC CT-3′, upstream primers (forward) 5′-GTGTA TGTCA GTGTG TTCCA GTCTT-3′ and (reverse) 5′-GCTGG GAGGG ATCTG CTCCT CTCTC-3′, and downstream primers (forward) 5′-GGGTT AAGGG GAGCA AAGTC AGAT-3′ and (reverse) 5′-CTGGG GAAAG GAGGC ACAAA GAAG-3′. PCR products were resolved by agarose-gel electrophoresis and visualized with ethidium bromide.ChIP assay was performed using the ChIP Assay Kit according to the manufacturer's instructions. Prx1 was cloned into the p3xFLAG-CMV-14 vector to create FLAG-Prx1 for use in the pull-down reactions. Antibodies used for ChIP assays were anti-HCC1, HSP70, Radixin, NF-κB p65, VDR, Moesin, TIF1α, FLAG, and normal mouse IgG. C3H10T1/2 cells were transfected with the Prx1-FLAG expression vector, treated with TNF-α (10 ng/mL), or control medium, and ChIP assay was done 24 hours later using 5 × 10MC3T3 cells were treated with control medium or TNF-α (10 ng/mL), as indicated in the figures. Cells were fixed in 95% ethanol for 10 minutes, followed by rinsing three times in PBS for 5 minutes each. Cells were blocked using 3% bovine serum albumin (BSA) in PBS (prepared from Blocker BSA in PBS) for 1 hour at room temperature. Prx1 antibody labeled with fluorescein isothiocyanate (FITC) was prepared according to kit instructions. Endogenous Prx1 was detected by incubating fixed cells with FITC–anti–Prx1 (1:50 in PBS) at 4°C overnight, followed by rinsing two times in PBS for 5 minutes each. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI), and cells were examined using a confocal microscope with a ×40 objective under oil immersion. Wavelengths used were 488 (FITC) and 350 nm (DAPI).Use of mice was approved by the institutional animal use committee of the VA Medical Center. Female TgTNF mice [B6.Cg(SJL)-Tg(TNF) N21 + ?] were purchased from Taconic . Tibias were harvested from 12-week-old mice expressing 3 to 5 pg/mL of human TNF-α or from wild-type littermates. Bones were cleaned, fixed in paraformaldehyde, and embedded in paraffin, and 5-µm cuts of the proximal tibia were made to include a portion of the cartilage, trabecular bone, and periosteal bone. Sections were processed for immunohistology using the antibody raised in our laboratory and described earlier.ANOVA was used to determine a statistical difference between multiple groups. Multiple comparisons between individual groups were done by the method of Tukey. Comparison between any one group and a common control was done by the method of Dunnett using Prism software .The TNF-responsive sequence from the Osx promoter, TTGGATCTGAGTGGGAACAAGAGTGAGCTG, was bound to sepharose beads and used as bait to identify the interacting nuclear protein complex in MC3T3 cells. MS identified fragments from 655 individual proteins. Since the probability of a true positive TNF-regulated protein increases with the number of identified fragments of that protein, further analysis was restricted to 62 proteins for which 2 or more fragments were detected and TNF modified detection of the protein. arrow) and confirmed the constitutive binding of moesin and TIF1β. ChIP assays were done to confirm binding of the selected proteins to the TNF-α repressor element of the Osx promoter. C3H10T1/2 cells were used for ChIP assays after treatment with TNF-α (10 ng/mL) or control medium. These experiments confirmed binding of moesin, TIF1β, and Prx1 to the region containing the TNF-α element. Osx transcription. Prx2 gene. The comparative structures of Prx1 isoforms and Prx2 are shown in Since TNF-α stimulated binding of Prx1, a factor previously described as a regulator of skeletogenesis, we focused on this factor as a potential mediator of TNF-α suppression of Treatment of undifferentiated MC3T3 cells with TNF-α stimulated the accumulation of Prx1 protein in the cytoplasm and nucleus within 15 minutes, as shown by fluorescent immunohistology A. This nOsx by silencing Prx1 with a short interfering RNA (si-Prx1) that inhibited expression of all Prx1 isoforms. Osx mRNA. Blockade of TNF-α inhibitory action by si-Prx1 was not observed for RUNX2 mRNA  that increased in intensity after 18 hours of TNF-α treatment . This complex contained Prx1 because the top band was supershifted by our Prx1 antibody (αPRX1#2) but not by a commercial antibody (αPRX#1) or a Prx2 antibody (αPRX2). Specificity of binding also was demonstrated by competition with 100X cold TNF element probe (TRE) but not by a control DNA sequence. Analysis of the Osx promoter sequence revealed a homologous homeobox-binding site just upstream of the 18-bp TNF-α response element, contiguous with a previously described RUNX2-binding site. Therefor32P-labeled TNF-α element, the recombinant proteins, and nuclear extract from TNF-α-treated C3H10T1/2 pluripotent mesenchymal cells. Prx1b and Prx2, but not Prx1a or the control reaction, readily bound the probe under these conditions. Binding of recombinant Prx1b required the inclusion of nuclear extract in the EMSA reaction, as shown in Binding to the promoter was evaluated further with recombinant Prx1a and Prx1b. Proteins were synthesized by in vitro transcription/translation using isoform-specific cDNA templates driven by the bacterial T7 promoter. These protein products or a control reaction supernatant was tested for binding to the TNF-α element of the Osx promoter. Osx mRNA levels by 50%, similar to the action of TNF-α., present in Prx1a, but not Prx1b, has been found to be an inhibitor of cription. The OAR sequence.Our prior studies of the Osx promoter revealed a MAPK/MEK/ERK1/2-dependent mechanism for TNF-α action. Although additional studies will be required to determine if TNF-α activation of Prx1b involves MAPK, experiments were reported to show MAPK/MEK/ERK1/2 and also AKT dependence for Prx action in amphibian regenerating limbs.TNF-α has a major role in the pathophysiology of skeletal disorders, including inflammatory arthritis, menopausal bone loss, and aging. These disorders share a reduced capacity of OBs to balance bone formation with resorption. Insufficient OB differentiation and function are likely reasons for this defect. We and others established TNF-α as an inhibitor of early commitment of OB precursors.,16,17,56


The role of autophagy in cell death is under considerable debate. The process of autophagy has been shown to lead to either cell survival or cell death depending on cell type and stimulus. In the present study, we determined the contribution of ERK1/2 signalling to autophagy and cell death induced by tumour necrosis factor-α (TNF) in MCF-7 breast cancer cells. Treatment of MCF-7 cells with TNF caused a time-dependent increase in ERK1/2 activity. There was an induction of autophagy and cleavage of caspase-7, -8, -9 and PARP. Pharmacological inhibition of ERK1/2 phosphorylation with U0126 or PD98059 resulted in a decrease in TNF-induced autophagy that was accompanied by an increase in cleavage of caspase-7, -8, -9 and PARP Furthermore, inhibition of ERK1/2 signalling resulted in decreased clonogenic capacity of MCF-7 cells. These data suggest that TNF-induces autophagy through ERK1/2 and that inhibition of autophagy increases cellular sensitivity to TNF. Macroautophagy (referred to hereafter as autophagy) or ‘self-eating’ is a mechanism of degradation and recycling of a cell's own components, including long-lived proteins and damaged organelles . Autophain vitro supporting a requirement for autophagy in the cell death response to nerve growth factor (NGF)-deprivation or cytosine arabinoside [There is a considerable debate regarding the role of autophagy in cell death , 5, 6. Tbinoside , serum abinoside , tumour binoside , caspasebinoside , brevinibinoside , etoposibinoside . In contbinoside . Furtherbinoside , starvatbinoside  and infebinoside . In addibinoside . Thus, aDrosophila salivary glands during development [Autophagy has been shown to precede apoptosis in human cervical cancer HeLa cells  and in Delopment , 19. Furelopment , furthervia ERK1/2 and inhibition of ERK1/2 enhances sensitivity of MCF-7 human breast cancer cells to TNF-induced cell death.Engagement of the receptor-mediated or extrinsic cell death pathway by ligands such as TNF and TNF-related apoptosis-inducing ligand (TRAIL) has been previously shown to induce autophagy. TRAIL induces autophagy in the lumen of MCF-10A cells grown in three-dimensional culture  and in pHuman recombinant TNF was purchased from R&D systems . Pharmacological inhibitors U0126 and PD98059 were purchased from EMD Biosciences . Monoclonal antibodies to phospho-ERK1/2 and GAPDH, and polyclonal antibodies to enhanced green fluorescent protein (EGFP) and caspase-9 were purchased from Santa Cruz Biotechnology, Inc. . Monoclonal antibody to caspase-8 was from Biosource, Invitrogen . Monoclonal antibodies to PARP, caspase-7 and ERK2 were purchased from BD Biosciences . The rabbit polyclonal antibody against LC-3 was kindly provided by Dr. T. Yoshimori  . Horsera2. MCF-7 cells were transfected with EGFP vector alone or LC3-EGFP construct performed using Fugene HD . Cells were selected in antibiotic (G418) to generate stable cell lines.MCF-7 cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated foetal bovine serum and 2 mM glutamine, and kept in a humidified incubator at 37°C with 95% air and 5% COEquivalent amounts of protein from total cellular extracts were electrophoresed by SDS-PAGE and transferred electrophoretically to poly(vinylidene difluoride) membrane. Immunoblot analyses were performed as described before .2 until there were at least 50 cells per colony in untreated cells. At the end of the incubation, cells were washed with phosphate-buffered saline (PBS) and incubated with a 0.25% crystal violet solution for 15 min. Following washes with PBS, colonies were counted using the colony-counting tool in the BioChemi System .Clonogenic assay was performed as described previously . Brieflyanova followed by Tukey's post hoc test. All analyses were performed using the VassarStats web site program available at: http://faculty.vassar.edu/lowry/VassarStats.html. Differences were considered significant at P< 0.05.Data from clonogenic assays were analysed by We have previously shown that TNF induces apoptosis in MCF-7 breast cancer cells , 35. In Myosin-associated protein1-light chain 3B (LC3) is the mammalian ortholog of yeast Atg8  and is iPE-conjugated LC3 is localized to autophagosomal membranes during autophagy . Thus, rLittle is known about the signalling pathways that regulate TNF-induced autophagy in breast cancer. We found that TNF activated ERK1/2 in a time-dependent manner  and was Signalling through the MAPK pathway has been associated with autophagy , 26, 38.We further determined the consequence of decreased autophagy and increased PARP cleavage on long-term cell survival. In clonogenic assays , we founvia ERK1/2 signalling was a protective event since a decrease in autophagy increased PARP cleavage and decreased clonogenic capacity of the cells.In the present study we have shown that TNF can activate both apoptosis and autophagy in MCF-7 breast cancer cells. Pharmacological inhibition of ERK1/2 was associated with a decrease in TNF-induced autophagy while it augmented TNF-mediated apoptosis. Furthermore, the induction of autophagy by TNF TNF induced LC3 processing as well as LC3 localization in autophagosomes in MCF-7 cells. Induction of autophagy by TNF has also been shown in lymphoblastic T cells , Ewing'sIt has been proposed that cells with compromised apoptotic machinery undergo autophagy more readily than cells with intact apoptotic capacity –44. In cvia the ERK1/2 pathway. The Ras-Raf-MAPK pathway, has emerged as a key regulator of autophagy [et al. (2007) showed that MEK inhibitors prevented curcumin-induced autophagy in glioma cells, suggesting that the activation of ERK is associated with the induction of autophagy [Little is known about signalling pathways that regulate TNF-mediated autophagy. We have demonstrated that TNF induces autophagy in MCF-7 cells utophagy , 27, 48.utophagy . In addiutophagy . Recentlutophagy .via the JNK pathway [There is some evidence that the JNK pathway can regulate autophagy in response to TNF  and thro pathway . Howeveret al. (1998) found that cells that were more sensitive to TNF-induced cell death underwent greater extent of autophagy [Our results suggest that inhibition of signalling through ERK1/2 enhanced TNF-induced cell death and reduced clonogenic capacity. In contrast, there is evidence that autophagy contributes to apoptotic cell death. For example, inhibition of autophagy in T-lymphoblastic cells  and in Eutophagy . In addiutophagy . Our resutophagy .Whether autophagy enhances or diminishes the apoptotic response is a much-debated issue. Indeed, understanding of the interplay between autophagy and apoptosis is critical if the autophagy pathway is to be targeted effectively in the treatment of diseases. Taken together, data from the present study demonstrate that inhibition of ERK1/2 signalling results in decreased autophagy in response to TNF. This was associated with increased sensitivity of MCF-7 cells to TNF-induced cell death and further disruption of their clonogenic capacity. This suggests that TNF-induced autophagy is, in fact, protective against cytotoxicity in MCF-7 cells. Pharmacological inhibition of the MAPK pathway may therefore prove to be an attractive option to enhance drug efficacy in cancer cells where autophagy is a survival mechanism.


Tumor necrosis factor-α (TNF-α) plays an essential role in the regulation of bone homeostasis in several chronic immune and inflammatory joint diseases, where inhibition of TNF has led to significant clinical improvement. However, TNF-activated pathways and mechanisms involved in bone remodeling remain unclear. So far, TNF-α was known as an inhibitor of osteoblast differentiation and an activator of osteoclastogenesis. Recent contradictory findings indicated that TNF-α can also activate osteoblastogenesis. The paradoxical role of TNF-α in bone homeostasis seems to depend on the concentration and the differentiation state of the cell type used as well as on the exposure time. This review aims to summarize the recent contradictory findings on the regulation of bone homeostasis by TNF-α at the isolated cell, whole bone, and whole body levels. In addition, the involvement of TNF-α in the bone remodeling imbalance is observed in inflammatory joint diseases including rheumatoid arthritis and ankylosing spondylitis, which are associated with bone destruction and ectopic calcified matrix formation, respectively. Both diseases are associated with systemic/vertebral osteoporosis. Tumor necrosis factor-α (TNF-α) is one of the several pro-inflammatory cytokines involved in bone remodeling. Classically, TNF-α plays a major role in the regulation of bone homeostasis by stimulating osteoclastogenesis and inhibiting osteoblast (OB) function. It is also involved in the pathogenesis of chronic inflammation in both mouse and human models. Based on these observations, treatments of several chronic inflammatory diseases such as Crohn’s disease, ulcerative colitis, and psoriasis target TNF-α. TNF-α is also associated with the pathogenesis of inflammatory joint diseases including rheumatoid arthritis (RA), which is characterized by massive juxta-articular bone destruction, and ankylosing spondylitis (AS), which is characterized by simultaneous bone destruction and excessive formation, respectively. However, opposite findings suggest that TNF-α may also induce osteogenic differentiation. This review summarizes the classical and paradoxical effects of TNF-α on bone homeostasis by focusing on osteoblastogenesis and osteoclastogenesis.The immune response is modulated by several inflammatory cytokines including TNF-α. TNF-α acts through several pathways including the activation of nuclear factor kappa-B (NF-κB) involved in inflammation and apoptosis . TNF-α iActivation of TNFR1 death domain initiates the apoptotic signaling through the activation of several factors such as caspase and NF-κB resulting in transcription of pro-inflammatory cytokines, chemokines, and anti-apoptotic molecules . Unlike In humans, TNF-α antagonists are clearly effective in a large number of conditions, as first shown in patients with RA , 10. HowBone structure is affected by several paracrine and endocrine factors including mechanical abrasion and serum electrolyte levels such as calcium and phosphorus. This remodeling is regulated by several cells such as osteoclasts (OCs), OBs, and osteocytes (OYs) . This coOsteoclasts are hematopoietic cells derived from the monocyte lineage. They undergo a series of differentiation steps to become mature bone-resorbing cells. Intracellular signaling cascade of transcriptional regulation of osteoclast-specific genes is activated by macrophage-colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL)  Figure . Upon diOsteoblasts are derived from pluripotent stem cells called “mesenchymal stem cells (MSCs)” capable of differentiating into adipocytes, chondrocytes, fibroblasts, skeletal muscle cells, and tendon cells . A numbeOsteocytes are derived from MSCs through OB differentiation. They are located within the bone matrix and represent 90–95% of all bone cells in adults . In addiin vitro studies have indicated an inhibitory effect of TNF-α on OB differentiation , which increase with estrogen deficiency, can induce OYs apoptosis . In a flin vitro demonstrated that they have specific characteristic osteoclastogenic properties. However, they exhibited osteoclastogenesis-related activity only in the presence of permissive levels of RANKL. TNF-α appears to have osteoclastogenic properties in the early stage of osteoclastogenesis when bone marrow-derived macrophages (BMMs) are at the stage of osteoclast precursor cells . The latter activates NF-κB, AP-1, and MAPKs signaling pathways, which leads to decreased bone formation.In human TNF transgenic mice, TNF-α induced DKK-1, a regulatory molecule of the Wnt pathway, which led to impaired local bone formation. In addition, blockade of DKK-1 interfered with local bone resorption by reducing OCs numbers in the joints of animal models with inflammatory arthritis , 63, 64.However, this observation was not clear in TNF-deficient mouse models, due to insignificant reduction in the clinical parameters of CIA and absence of major histological and morphological abnormalities in the joints. Moreover, some mice exhibited severe disease in the absence of TNF, suggesting the contribution of other cytokines such as IL-1 –68.Tumor necrosis factor-α has been associated with an increasing number of chronic inflammatory diseases. Among these diseases, the two most common inflammatory joint diseases RA and AS will be discussed. RA causes massive destruction of juxta-articular and systemic bone while AS leads to the concomitant bone destruction and ectopic bone formation  because of inflammation of tendon/ligament insertions. Thus, patients with AS experience stiffness of the spine known as ankylosis, and functional disability –71. InhiRheumatoid arthritis is a chronic disease characterized by joint inflammation leading to destruction of cartilage and bone . In RA, The pro-inflammatory influence of TNF-α in RA acts at several levels. It induces other pro-inflammatory cytokines such as IL-1 and IL-17 and regulates osteoclastogenesis via several mechanisms. TNF-α accelerates cartilage destruction through inducing the production of matrix metalloproteinases (MMPs) and aggrecanases (ADAMTSs), which are enzymes that degrade components of the cartilage extracellular matrix. As an example, in human RA patients and in the human TNF-α transgenic mouse model, TNF-α was shown to regulate MMP3 and 13 by the small ubiquitin-like modifier-1 (SUMO-1) through NF-κB, which led to bone destruction . In addiin vivo inhibition of TNF in AS patients. TNF blockers reduce signs and symptoms of AS, which translates into better physical function and quality of life. However, little or no effect on structural remodeling is achieved (Ankylosing spondylitis is the second most common form of chronic inflammatory arthritis. The unique hallmark of the disease is pathologic new bone formation . AS and achieved , 90.In AS, relevant human samples are not readily available even so TNF expression has been shown at the site of sacroiliitis. Thus, most observations are only made in rodent models. Recently, mice that specifically overexpress the transmembrane but not the soluble form of TNF-α, so called tmTNF transgenic mice, developed clinically spinal abnormalities similar to those in AS . These mOsteoporosis is characterized by a decrease in bone mass and density leading to bone weakness and prone to fracture. In RA patients, several studies have shown an increased incidence of osteoporosis. This could be due to the highly systemic inflammatory environment observed in these patients and the use of glucocorticoid treatment, which can affect bone remodeling , 94. Desin vitro models and were concentration- and time-dependent, and cell-type specific. Despite the fact that TNF-α levels are high in both RA and AS, the bone dynamics are different. In RA, the dynamics are shifted toward bone destruction, combined with inhibition of specific osteogenic genes. In AS, systemic bone destruction is accompanied by ectopic bone formation at sites where OCs and OBs are disconnected. Future research should focus on the delineation of the molecular pathways used by this mediator and understand the conditions affecting and the purpose of this dual role in bone remodeling.Tumor necrosis factor-α has an important role in the regulation of bone homeostasis via activation of complex signaling pathways leading to gene transcription of several regulators of bone homeostasis. The classical view remains that TNF-α is an established inducer of osteoclastogenesis and inhibitor of osteoblastogenesis. To add further complexity, it was recently discovered that TNF-α also possessed osteogenic differentiation effects. Most of these paradoxical effects were only described in The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Herein, we explored the effects and mechanisms of sodium hydrosulfide  on tumor necrosis factor (TNF)-α-induced human umbilical vein endothelial cells (HUVEC) dysfunction.Hydrogen sulfide (HApplication of NaHS concentration-dependently suppressed TNF-α-induced mRNA and proteins expressions of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), mRNA expression of P-selectin and E-selectin as well as U937 monocytes adhesion to HUVEC. Western blot analysis revealed that the expression of the cytoprotective enzyme, heme oxygenase-1 (HO-1), was induced and coincident with the anti-inflammatory action of NaHS. Furthermore, TNF-α-induced NF-κB activation assessed by IκBα degradation and p65 phosphorylation and nuclear translocation and ROS production were diminished in cells subjected to treatment with NaHS.2S can exert an anti-inflammatory effect in endothelial cells through a mechanism that involves the up-regulation of HO-1.H Endothelial dysfunction elicited by inflammatory cytokines is regarded as a key event in the pathogenesis of cardiovascular disorders Several studies indicated expression of adhesion molecules on endothelial cells induced by tumor necrosis factor-α (TNF-α) is associated with activation of multiple signal transduction pathways, including mitogen-activated protein kinases (MAPK) and nuclear factor-κB (NF-κB) 2S), a colorless gas with a characteristic rotten-egg odor, has traditionally been considered to be a toxic environmental pollutant. More recently, H2S has been identified as the third, physiologically relevant, gaseous signaling molecule with a diverse physiological profile 2S production has been attributed to three key enzymes cystathionine γ-lyase 2S appears to confer cytoprotection via multiple mechanisms including anti-oxidant and anti-inflammatory effects 2S acts as an endogenous scavenger for ROS and reactive nitrogen species 2S has been implicated to play a pro-inflammatory role in systemic inflammation 2S is a potent anti-inflammatory molecule in various models 2S in endothelial cells has not yet been clarified. Here, our present work investigated if H2S exerts anti-inflammatory and thereby potential anti-atherogenic properties in endothelial cells through inhibition of pro-inflammatory processes, such as the expression of adhesion molecules, intracellular ROS production. In addition, the underlying mechanisms and intracellular signaling pathways affected by H2S in TNF-α-stimulated endothelial cells were investigated.Hydrogen sulfide (HThe cytotoxicity experiments of NaHS in this study were performed at 10–100 µM concentration. Non-cytotoxic effect of NaHS was observed at the dosage used in this study (data not shown).P>0.05). These results suggested that ROS is involved in the binding of U937 cells to TNF-α-stimulated endothelial cells.We first investigated the effect of NaHS on the adhesion of U937 cells to TNF-α-activated endothelial cells, a critical step in vascular inflammation. As shown in P>0.05).The effects of NaHS on TNF-α-induced mRNA levels of adhesion molecules by HUVEC were studied by quantitative real-time RT-PCR. Resting HUVEC showed a low constitutive transcription of adhesion molecules, while TNF-α caused a significant increase in mRNA levels of E-selectin, P-selectin, VCAM-1, and ICAM-1 in HUVEC after 4 h of incubation. Treatment of cells with NaHS (10-100 µM) for 30 min resulted in concentration-dependent decreases in TNF-α-induced mRNA levels of E-selectin , P-selecAs the expression of adhesion molecules on endothelial cells is a prerequisite for adhesion of leukocytes, we investigated the effect of NaHS on TNF-α-induced ICAM-1 and VCAM-1 expression. Western blot analysis of cell lysates showed that levels of VCAM-1 and ICAM-1 were very low in unstimulated HUVEC, but were significantly increased by TNF-α treatment, NaHS attenuated TNF-α-induced ICAM-1 expression only at higher concentration (100 µM) , while NThe effect of NaHS on HO-1 expression was initially explored in cultured HUVEC, 2S on TNF-α-induced intracellular ROS production, HUVEC were labeled with a cell-permeable fluorescent dye H2DCF-DA and analyzed by spectrofluorometer or fluorescence microscope. Stimulation with TNF-α resulted in a great increase in the amount of intracellular ROS generation in HUVEC compared with unstimulated cells (P>0.05). These results strongly suggested that scavenging ROS by H2S may be responsible for inhibition in the binding of U937 cells to TNF-α-stimulated endothelial cells.To confirm whether the inhibitory effect of Hed cells . Howevered cells . MeanwhiActivation MAPK signaling pathway induced by TNF-α plays an important role in the regulation of adhesion molecules expression. In unstimulated cells, there was almost no detectable phosphorylation of p38, ERK1/2, or JNK1/2 in HUVEC. TNF-α caused a rapid phosphorylation of MAPK within 5 min, with phosphorylation peaking at 15 min and followed by dropping to normal level . Based oTo examine whether MAPK activation was involved in the regulation of inflammatory response by NaHS in TNF-α-stimulated HUVEC, phosphorylation of MAPK  were analyzed by Western blot. As shown in Translocation of NF-κB from cytoplasm to the nucleus is preceded by the phosphorylation and subsequent degradation of IκBα. To determine the effect of NaHS on TNF-α-induced IκBα degradation, total cell lysate was prepared from the TNF-α with or without NaHS-treated cells. Using Western blot analysis, we demonstrated that the degradation of IκBα took place in a time dependent manner after the TNF-α induction . As showSer536 in the TNF-α-activated HUVEC, Western blot analysis was performed. As shown in Ser536 in HUVEC. NaHS markedly reduced TNF-α-induced phosphorylation of NF-κB p65Ser 536 in a concentration dependent manner.The phosphorylation of NF-κB p65 subunit, particular on serine residues 536 in the C-terminal transactivation domain, plays an important role in regulation transcription of adhesion molecules following exposure to inflammatory stimuli We next asked whether NaHS might play a role in the regulation of nuclear NF-κB p65 translocation in TNF-α-stimulated HUVEC. The translocation of NF-κB p65 from the cytoplasm to the nucleus was visualized by Western blot and immunofluorescence . TNF-α r2S, at the dosage used in this study, attenuated TNF-α-induced endothelial dysfunction in vitro. Our major findings showed that exogenous H2S blocked the adhesion of U937 cells to TNF-α-activated HUVEC by inhibiting expression of adhesion molecules; suppressed the TNF-α-induced activation of NF-κB by inhibiting degradation of IκBα and activation of p38 signaling pathway; eliminated TNF-α-induced intracellular ROS production; and up-regulated HO-1 expression in HUVEC.Inflammation contributes to the pathogenesis of cardiovascular disease and elevated level of pro-inflammatory cytokine TNF-α is associated with endothelial dysfunction 2S has multiple positive physiological functions, but the role of H2S during systemic inflammatory diseases is still a matter of debate or may be double-edged. NaHS was used as a H2S donor, because it can dissolves into Na+ and HS− in solution, HS− is released and forms H2S with H+. This provides a solution of H2S at a concentration that is about 33% of the original concentration of NaHS 2S level in cardiovascular disease 2S has a protective effect against atherosclerosis in apoE−/− mice and attenuated TNF-α-induced ICAM-1 expression in HUVEC 2S levels in the 50–150 µM range 2S may function as a modulator of endothelial function at the relevant physiological concentrations (10–100 µM).As the third gaseous mediator H2S might exert anti-inflammatory effect via multiple mechanisms such as upregulation of antioxidant defense 2S exert their anti-oxidative effects by inhibiting ROS production induced by cytokines or hydrogen peroxide in mouse pancreatic β-cells 2S inhibited TNF-α-induced expression of ICAM-1, as previously reported by Wang 2S has been demonstrated to play a proinflammatory role in various disease states 2S donor used or a different inflammatory model.It is well known that adhesion molecules are strong predictors of atherosclerotic lesion development and future cardiovascular events 2S to coordinate to other thiol-containing protein, including a number of redox-sensitive transcription factors and kinases, which is a crucial modulator of the expression of antioxidant genes, including HO-1. We also suggested that HO-1 induction by NaHS might contribute to its anti-inflammatory action. Because the expression of HO-1 was induced concomitantly with the attenuation of expression of adhesion molecules and the binding of U937 cells to TNF-α-stimulated HUVEC exerted by NaHS. The result is consistent with the report that overexpression of HO-1 prevented adhesion molecules expression and leukocytes to activated endothelial cells HO-1 is the inducible isoform of the first and rate-controlling enzyme of heme degradation and plays a central role in the regulation of inflammatory reaction via its products bilirubin and carbon monoxide in a variety of experimental systems 2S can attenuate TNF-α-induced NF-κB activation, as previously reported MAPK and NF-κB are key players in intracellular signaling pathways in response to inflammatory stimuli and required for adhesion molecules expression 2S that improved TNF-α-induced endothelial dysfunction. These findings suggested that H2S release agents could represent a promising approach for the treatment of inflammatory vascular diseases.In summary and conclusion, we demonstrated that NaHS attenuated expression of adhesion molecules and monocyte adhesion to endothelial cells, and decreased intracellular ROS production in TNF-α-stimulated endothelial cells. Remarkably, this anti-inflammatory effect is primarily achieved by the inhibition of NF-κB and p38 signaling pathways, and by the upregulation of HO-1 expression. We, thus, concluded that the inhibition of NF-κB and p38 signaling pathways, adhesion molecules, and modulation of cellular redox balance might be one of the important mechanisms of H2DCF-DA) was from Molecular probes . NaHS, N-acetyl-L-cysteine (NAC), and dexamethasone (Dex) were purchased from Sigma-Aldrich . NaHS has been well established as a reliable donor of H2S in culture media DMEM, RPMI-1640, and fetal bovine serum (FBS) were from GIBCO-BRL (USA). Recombinant human TNF-α was purchased from Millpore . 2′,7′-dichlorodihydrofluorescein diacetate  were maintained in RPMI-1640 containing 1800 mg/L NaHCO3, 4500 mg/L glucose, and 110 mg/L sodium pyruvate, supplemented with 10% FBS, 100 U/ml penicilin and 100 µg/ml streptomycin at 37°C in a humidified atmosphere with 5% CO2.HUVEC  were grown in DMEM supplemented with 1800 mg/L NaHCOµM) for 30 min before stimulating with TNF-α (10 ng/ml) for various periods: 15 min for measurement of MAPK  phosphorylation, IκBα protein, and NF-κB p65ser536 phosphorylation. 1 h for measurement of intracellular ROS production and NF-κB p65 translocation; 4 h for measurement of mRNA levels; 6 h for measurement of proteins expression of adhesion molecules, HO-1, and adhesion assay.For experiments, HUVEC were grown to confluence in 6-well plates, 24-well plates, or 60 mm dishes . Cells were serum-starved for 12 h, to assess the effect of TNF-α or NaHS on MAPK phosphorylation, cells were incubated with TNF-α (10 ng/ml) for different periods . In some experiments, cells were pre-incubated with or without NAC (5 mM), Dex (10 µM), or NaHS  according to the manufacturer's directions. real-time RT-PCR conditions include denaturation (95°C for 30 s); annealing (60°C for 30 s); number of cycles (40) and were performed using iQ5 real-time RT-PCR detection System  in a total volume of 25 µl reaction mixture containing 2 µl cDNA, 12.5 µl 2× SYBR Green 1 Master Mix (Takara), and 1 µl of each primer. GAPDH was used as an internal control to compare the amount of total mRNA of each sample. The primers used in this experiment were indicated in Total RNA was extracted from HUVEC with TRIzol Reagent  following the manufacturer's instructions. Total RNA (2 µg) of each sample was reverse-transcribed into cDNA and amplified using a PrimeScriptFor whole cell extraction, cells were washed twice with ice-cold PBS and lyzed in RIPA buffer with protease & phosphatase inhibitor. After centrifugation , samples were prepared for Western blot analysis.NE-PER@ Nuclear and Cytoplasmic Extraction Reagents  according to manufacturer's instructions.For preparation of cytoplasmic and nuclear fraction, HUVEC were pretreated with NaHS (100 µM), NAC (5 mM), or Dex(10 µM) for 30 min, and then stimulated with TNF-α for 1 h. Nuclear and cytoplasmic proteins of HUVEC were extracted using the 202/Tyr204), JNK1/2, p-JNK1/2 (Thr183/Tyr185), p38, p-p38 (Thr180/Tyr182), NF-κB p65, p-NF-κB p65 (Ser536), IκBα (1∶1000) ; ICAM-1 (1∶500), VCAM-1 (1∶500), β-Tubulin (1∶2000), β-actin (1∶500), GAPDH (1∶2000), or HO-1 (1∶2000) . After incubation with appropriate secondary antibodies for 1 h at room temperature, proteins were visualized by enhanced chemoluminescence with a camera-based imaging system . The density of the signals was quantified with the AlphaEase software.Equal amounts (30 µg) of proteins were separated on 8-10 % sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinyl difluoride membrane . After being blocked with 5% nonfat dry milk, membranes were incubated overnight at 4°C with primary antibodies against ERK1/2, phosphorylated (p)-ERK1/2  and Alexa Fluor 488 conjugated goat anti-rabbit IgG , and counterstained for nuclei with DAPI. Immunofluorescence was visualized using a fluorescent microscope (Carl Zeiss Inc.). The results were based on three independent analyses.2DCF-DA, was used to measure the intracellular generation of ROS by TNF-α 2DCF-DA for 30 min. Then stimulated with TNF-α (10 ng/ml) for 1 h, and the fluorescence intensity was measured at an excitation and emission wavelength of 485 nm and 530 nm, respectively, using a fluorescence spectrophotometer  or a fluorescence microscope (Carl Zeiss).The fluorescent probe, HP<0.05.Data are presented as mean ± S.E.M. Differences between mean values of multiple groups were analyzed by one-way analysis of variance with Dunnett's test for post hoc comparisons. Statistical significance was considered at


Immunoglobulin new antigen receptors (IgNAR) are a unique subset of antibodies consisting of five constant (CNAR) and one variable domains (VNAR). VNAR domains are the smallest, naturally occurring, antibody-based immune recognition units, having potential use as therapy.In sepsis, tumor necrosis factor (TNF) is the key factor triggering respiratory burst, tissue injury and disseminated coagulation. Anti-TNF strategies based on monoclonal antibodies or F(ab’)VNAR on survival in an experimental model of endotoxic shock. Also, mRNA expression and serum protein of several inflammatory molecules were measured.Our aim was to explore the impact of an anti-TNF VNAR domains, F(ab’)2 antibody fragments, or saline solution 15 minutes before, 2 h and 24 h after lethal dose100 (LD100) LPS administration. TNF blockade with either VNAR domains or F(ab’)2 fragments were associated with lower mortality  compared to LD100. Challenge with LPS induced significant production of serum TNF and interleukins -10 and -6 at 3 h. After that, significant reduction of IL-6 at 24 h (vs 3 h) was shown only in the VNAR group. Nitrites level also increased in response to LPS.Endotoxic shock was induced by lipopolysaccharide (LPS) in male Balb/c mice. Animals were treated with anti-TNF 2 fragments than in those with VNAR therapy. With regard to IL-6, gene expression was increased at 3 h in all groups. TNF blockade was associated with rapid and sustained suppression of IL-6 expression, even more evident in the VNAR group. Finally, expression of inducible-nitric oxide synthase (iNOS) increased in response to LPS at 3 h, but this was decreased at 24 h only in the anti-TNF VNAR group.In liver, TNF and IL-10 mRNA expression showed a pro-inflammatory imbalance in response to LPS. Blocking TNF was associated with a shift towards an anti-inflammatory status; however, polarization was more pronounced in animals receiving F(ab’)VNAR single domains improved survival in a murine model of endotoxic shock. Protection was associated with regulation in the TNF/IL-10 balance, attenuation of IL-6 and iNOS gene expression in the liver as well as decreased serum IL-6 concentration.Anti-TNF  Sepsis is a life-threatening condition which results from an exacerbated inflammatory process caused by complex interactions between the innate immune system and either bacterial sources of infection or their circulating antigens, mainly lipopolysaccharides (LPS). Circulating LPS is engaged by Toll-like receptor 4 (TLR4) on the surface of monocytes and macrophages, which in turn result in cell activation and early production of large amounts of inflammatory cytokines ,2. Tumor2 fragments [Elucidation of pathophysiological events that underlie sepsis has led to the development of molecules targeting these pathways. Major strategies to block TNF have included the use of anti-TNF monoclonal antibodies, soluble TNF receptors with IgG chimeric protein, and anti-TNF F(ab’)ragments -8. InterVNAR) and five constant (CNAR) domains. Single VNAR domains are candidates to be the smallest, naturally occurring, antibody-based immune recognition units [VNAR domains have been suggested as a highly effective set of molecules capable to access antigenic sites rarely targeted by conventional antibody-based strategies, positioning them as attractive candidates for therapy [VNAR single domain directed against TNF.Immunoglobulin new antigen receptors (IgNAR) are a unique subset of antibodies found in sharks. It consists of homodimers of polypeptide chains, each comprising a single variable 2 fragments reduced mortality as early as 24 h and continued until 96 h surveillance. VNAR domains were associated with lower mortality compared with F(ab’)2 fragments, although this was not significant , however both groups were significantly protected vs LD100 treatment (p < 0.001 and 0.05 respectively).Survival of mice was documented for up to four days after induction of endotoxemia Figure . All ani2 group (411 ± 79 pg/mL), and VNAR group (947 ± 324 pg/mL) compared to animals with neither administration of LPS nor anti-TNF . Nevertheless, F(ab’)2 fragments induced a persistent decrease of TNF at 24 h (38 ± 38 pg/mL) and 48h (0 pg/mL), while VNAR domains produced a transitory decrease at 24 h , with a subsequent mild increase at 48 h (121 ± 121 pg/mL). Endotoxemic shock was also associated with an early raise of IL-10  and with VNAR groups (418 ± 119 pg/mL). After 24 h both anti-TNF treatments induced a similar decline in the IL-10 concentration.Induction of endotoxic shock produced a significant increase in the serum concentration (mean ± standard error) of TNF at 3 h after LPS administration Figure , panel A2 group from 3 h to 48 h of surveillance, VNAR treated animals showed a pro-inflammatory response characterized by high TNF and low IL-10 serum concentrations.TNF/IL-10 ratio was considered to represent antagonistic inflammatory responses in endotoxic shock . While a2 group showed a gradual decrement (from 13392 ± 1749 to 7919 ± 1991 pg/mL at 24 h), while this decline was abrupt in the VNAR group . IL-6 levels were undetectable at 48 h in all groups.Serum concentrations (mean ± standard error) of IL-6 were similar at 3 h in all endotoxemic shock groups Figure , panel C100 (1256 ± 262 μM/mL)(p < 0.05) and F(ab)2 (1581 ± 438 μM/mL) (p < 0.001) groups vs normal group (206 ± 74 μM/mL), but not in VNAR group (592 ± 144 μM/mL) 2 fragments group (from 1581 ± 438 to 4000 ± 657 μM/mL).Nitrites concentration (mean ± standard error) in sera was found to be elevated at 3h in LD100 of LPS induced a predominant pro-inflammatory response, TNF blockade with either molecule was associated with an anti-inflammatory predominance. However, subtle differences in the TNF/IL-10 balance were found, because polarization to an anti-inflammatory status was more pronounced in the animals receiving F(ab’)2 fragments than in those with VNAR therapy.Relative mRNA expression of TNF and IL-10 in liver increased at 3 h as a result of LPS administration 2 to suppress IL-6 mRNA expression at 24 h  and 48 h (5.69 versus 24.20 p < 0.05) after induction of endotoxemia.Interleukin-6 gene expression in liver increased early (3 h) after LPS challenge in all groups Figure , panel FVNAR group this reduction was found to be statistically significant.Finally, expression of mRNA inducible-nitric oxide synthase (iNOS) in liver was similar between all groups at 3 h  show a mean serum half-life of 2 hrs [VNAR (14 KD) single domains as little as 3 min to 1.5 hrs [In the present study, anti-TNF administration was not associated with early (3 h) depletion of serum TNF levels; on the contrary, TNF was detected in sera from animals in all groups. In contrast, serum TNF declined to almost undetectable levels at 24 h and 48 h after administration of either F(ab’)ntration ,16. A pontration , mean seof 2 hrs , and VNA 1.5 hrs .VNAR single domains, they may arrive to the liver and, possibly, deeply penetrate in hepatic tissue [The liver plays key physiological roles including blood filtering of toxins as well as inactivation and clearing of bacterial antigens and products . Thus, tc tissue , which uknockout animals are associated with increased tissue injury [VNAR domains compared to F(ab’)2 fragments. This could be associated with the trend for higher survival observed in the group on VNAR therapy. In support to this notion, we have previously described that regulation in the IL-1β/IL-10 balance is associated with protection against lethality in a sepsis model of cecal ligation and puncture [The protective role of IL-10 in animal sepsis may be due to its antagonistic effect on the production and overall functioning of TNF -25. In te injury ,27 and he injury ,29. In opuncture . The preTumor necrosis factor directly influences the production of IL-6 and iNOS -33 and i2- concentrations [VNAR domains as an alternative approach in the treatment of sepsis, due to its attenuating effects on the inflammatory response showed in liver; however these must be further studied in more aggressive models such as the polymicrobial sepsis induced by cecal ligation and puncture. Related to the latter study, a recent rat gut model of indomethacin-induced jejunoileitis showed that anti-TNF-α monoclonal antibody reduced iNOS expression and IL-1 beta, the latter two thought to be key mediators of inflammatory bowel disease [In addition, protection of liver seems to be critical to obtain beneficial outcomes in sepsis. In a recent study performed in rats with polymicrobial sepsis, treatment with hyperoncotic albumin attenuates hepatic injury in association with reduced plasma levels of IL-1β, IL-6, liver enzymes, and Otrations . These r disease .VNAR single domains are a novel strategy useful to improve survival in a murine model of endotoxic shock, with efficacy similar to that observed with the use of anti-TNF F(ab’)2 fragments. Protection against lethality was associated with regulation in the TNF/IL-10 inflammatory balance, attenuation of IL-6 and iNOS liver expression, and decreased IL-6 serum concentration.Anti-TNF VNAR domains was performed as previously described [Heterodontus francisci was repeatedly immunized (intravenous route) with human recombinant TNF . After immunization, RNA from the spleen was obtained and used to amplify variable genes through polymerase chain reaction (PCR), and libraries were generated. A specific clone was obtained by phage display technique and cultures of Pichia pastoris were used to express it, in accordance with the Easy Select Pichia Expression Manual ; pPICZαAvector and X-33 strain were used. vNAR single domains were isolated and purified in the Centro de Investigación Científica y de Educación Superior de Ensenada (Mexico).Isolation of anti-TNF escribed ,40. In bad libitum . Initially, independent experiments were performed to investigate the Lethal Dose 100 (LD100), which was found to be 20 mg/kg of LPS serotype O55:B5  diluted in 100 μL 0.9% saline solution, administered by intraperitoneal (IP) injection in a single dose. Similarly, a pilot assay was performed to investigate the dose of anti-TNF VNAR domains useful to improve survival in the murine model of endotoxemic shock, doses of 0.01, 0.1 and 1 mg/kg of VNAR anti-TNF were tested, the latter dose was the only one that induced protection. To compare active treatment with anti-TNF VNAR domains, we used anti-TNF F(ab’)2 antibody fragments . We used the same dose of the latter antibody (1 mg/kg) since it had previously shown protective activity in the cecal ligation and puncture model.Protocol was approved by the ethics committee at the Instituto Nacional de Cardiología Ignacio Chávez (Protocol number 11–726). Male Balb/c mice, classified as an inbred based on 20 or more successive brother-sister matings , 8–12 we100 LPS in 100 μL 0.9% saline solution by IP injection, these mice also received 100 μl of 0.9% saline solution by IP injection, 15 minutes before as well as at 2 and 24 h after LD100 LPS was administrated; (B) 1 mg/kg anti-TNF VNAR domains diluted in 100 μl 0.9% saline solution by IP injection, 15 minutes before as well as at 2 and 24h after LD100 LPS was administrated; (C) 1 mg/kg anti-TNF F(ab’)2 antibody fragments diluted in 100 μL 0.9% saline solution by IP injection administrated in a similar time schedule than VNAR domains; (D) 100 μl 0.9% saline solution IP in the same time schedule as the other groups; these mice received neither LPS nor anti-TNF treatment.Mice (20 per group) were allocated in groups to receive (A) LD2-) by means of a Griess reagent system . All assays were performed according to manufacturer.Using another batch of mice, blood from 10 anesthetized animals for each group and time was obtained by cardiac puncture at 3, 24 and 48 h after induction of endotoxemia. Serum TNF, IL-6 and IL-10 concentrations were measured by enzyme-linked immunosorbent assays . In addition, non-hemolysed serum was used to measure nitrites . RNA integrity was assessed by agarose gel stained with ethidium bromide and purity was determined by spectrophotometer (260/280 > 1.8); 2 micrograms of total RNA were retro-transcribed using random primers in a 20 μl reaction with the Transcriptor First cDNA synthesis kit . One microliter of cDNA was amplified by qPCR using the LightCycler 2.0 with LightCyclerTaqMan Master Mix (Roche) and PCR primers in combination with LNA hydrolysis probes designed with the probe finder software v. 2.45 from the Universal Probe Library Mouse Set (Roche).PCR was performed using the following primers: Inducible nitric oxide synthase 2 (NM_010927.3), forward 5′-gggctgtcacggagatca-′3, reverse 5′-ccatgatggtcacattctgc-′3; IL-6 (NM_031168.1), forward 5′-gctaccaaactggatataatcagga-′3, reverse 5′-ccaggtagctatggtactccagaa-′3; IL-10 (NM_007393.3), forward 5′-cagagccacatgctcctaga-′3, reverse 5′-gtccagctggtcctttgttt-′3; TNF (NM_013693.2), forward 5′-tcttctcattcctgcttgtgg-′3, reverse 5′-ggtctgggccatagaactga-′3; and Actb (NM_007393.3), forward 5′-ctaaggccaaccgtgaaaag-′3, reverse 5′-accagaggcatacagggaca-′3 as a constitutive gen.-ΔΔCt. Assays were tested for linearity and reproducibility (variation coefficients <10%).Relative expression of each gene was calculated according to 2Results are expressed as proportions or mean ± standard deviation. Comparison of survival curves was performed using the Mantel-Haenzel log-rank test. Differences between two independent groups were calculated by Mann–Whitney tests, while those comparisons including more than two groups were performed by the Kruskal-Wallis test (Dunn’s post test). All analyses were 2-tailed and a p < 0.05 value was used for significance. The GraphPad Prism v 4.02 statistical software  was used.IgNAR: Immunoglobulin new antigen receptors; IL: Interleukin; iNOS: Inducible nitric oxide synthase; LD100: Lethal dose 100; LPS: Lipopolysaccharide; TLR4: Toll-like receptor 4; TNF: Tumor necrosis factor; VNAR: Single variable domain.FS-M, YY, IA, LB and LMA-G declare they have no competing interest. RM-V and RB received a research support by Laboratorios Silanes-Fundación Mexicana para la Salud. MTM-G, WJG-U and JFP-S were employed by Laboratorios Silanes at the time of the manuscript was written. TAC-V, ES-C and AFL-N received a grant support by Laboratorios Silanes. Funding source was provided by Laboratorios Silanes and Fundación Mexicana para la Salud with the support fund number 471.VNAR. MTM-G, WJG-U, JFP-S contributed in the isolation, characterization, and production of anti-TNF VNAR. All authors reviewed and approved the final manuscript.RM-V and RB contributed to the conception and design of the study, the interpretation of data and the elaboration of the manuscript. RM-V, BL, YY, AI, FS-M and LMA-G contributed with the development of experiments and in the interpretation of data. TAC-V, ES-C, WJG-U, AFL-N contributed in the isolation and production of anti-TNF