TNF Synthesis Research Articles

ZnPP-laden nanoparticles improve glucose homeostasis and chronic inflammation during obesity.

Chronic inflammation plays a pivotal role in the development of Type 2 diabetes mellitus (T2DM). Previous studies have shown that haem oxygenase-1 (HO-1) plays a proinflammatory role during metabolic stress, suggesting that HO-1 inhibition could be an effective strategy to treat T2DM. However, the application of HO-1 inhibitors is restricted due to solubility-limited bioavailability. In this study, we encapsulated the HO-1 inhibitor, zinc protoporphyrin IX (ZnPP), within nanoparticles and investigated their role in regulating glucose homeostasis and chronic inflammation during obesity. We delivered DMSO-dissolved ZnPP (DMSO-ZnPP) and ZnPP-laden nanoparticles (Nano-ZnPP) to diet-induced obese male mice for 6weeks. Glucose and insulin tolerance tests were carried out, liver and adipose tissue gene expression profiles analysed, and systemic inflammation analysed using flow cytometry. Nanoparticles significantly increased the delivery efficiency of ZnPP in both cells and mice. In mice with diet-induced obesity, inhibition of HO-1 by Nano-ZnPP significantly decreased adiposity, increased insulin sensitivity, and improved glucose tolerance. Moreover, Nano-ZnPP treatment attenuated both local and systemic inflammatory levels during obesity. Mechanistically, Nano-ZnPP significantly attenuated glucagon, TNF, and fatty acid synthesis signalling pathways in the liver. In white adipose tissue, the oxidative phosphorylation signalling pathway was enhanced and the inflammation signalling pathway diminished by Nano-ZnPP. Our results show that Nano-ZnPP has better effects on the improvement of glucose homeostasis and attenuation of chronic inflammation, than those of DMSO-dissolved ZnPP. These findings indicate that ZnPP-laden nanoparticles are potential therapeutic agents for treating T2DM.

1508-P: ZnPP-Laden Nanoparticles Improve Glucose Homeostasis and Chronic Inflammation during Obesity

Type 2 diabetes mellitus (T2DM) characterized by insulin resistance has become a serious health threat. Chronic inflammation is believed to play a pivotal role in the development of T2DM. Previous study has shown that HO-1 plays an anti-inflammatory role, suggesting HO-1 inhibition could be an effective strategy to treat T2DM. However, application of HO-1 inhibitors is restricted due to their low solubility. In this study, we detected the role of HO-1 inhibitor, ZnPP, in regulating glucose homeostasis and inflammation during obesity. We encapsulated ZnPP with nanoparticles to increase its water solubility and delivered ZnPP-laden nanoparticles (Nano-ZnPP) to the obese mice. We found that inhibition of HO-1 by Nano-ZnPP significantly decreases adiposity, increases insulin resistance, and improves glucose tolerance in diet-induced obesity mice. Moreover, Nano-ZnPP treatment attenuated both local and systematical inflammatory levels during obesity. Mechanistically, Nano-ZnPP significantly attenuated glucagon, TNF, and fatty acid synthesis signaling pathway in liver. In white adipose tissue, oxidative phosphorylation signaling pathway was enhanced and inflammation signaling pathway was diminished by Nano-ZnPP. Our results showed that Nano-ZnPP has a better effect on improving glucose homeostasis and attenuating chronic inflammation, as compared to DMSO-dissolved ZnPP. These results indicate that ZnPP-laden nanoparticles could be a potential therapeutic agent to treat T2DM. Disclosure W.Yang: None. Y.Sun: None. S.Guo: None. M.Arora: None. H.Han: None. W.Jiang: None. D.Kim: None. W.Ai: None. Q.Pan: None. M.Kumar: None. W.Brashear: None. Funding National Institutes of Health (R01DK095118, R01DK120968, R01DK118334, R01AG064869)

TAAR1 Regulates Purinergic-induced TNF Secretion from Peripheral, But Not CNS-resident, Macrophages.

Trace amine-associated receptor 1 (TAAR1) is an established neuroregulatory G protein-coupled receptor with recent studies suggesting additional functions related to immunomodulation. Our lab has previously investigated TAAR1 expression within cells of the innate immune system and herein we aim to further elucidate TAAR1 function in both peripherally-derived and CNS-resident macrophages. The selective TAAR1 agonist RO5256390 was used in combination with common damage associated molecular patterns (ATP and ADP) to observe the effect of TAAR1 agonism on modulating cytokine secretion and metabolic profiles. In mouse bone-marrow derived macrophages, TAAR1 agonism inhibited TNF secretion following ATP stimulation, which appeared to be downstream of an associated pro-inflammatory shift in metabolic profile and transcriptional regulation of TNF synthesis. In contrast, TAAR1 agonism had no effect on ADP-induced TNF and IL-6 secretion in mouse microglia in either the presence or absence of astrocytes. In summary, we report a novel interaction between TAAR1 and purinergic signaling in peripherally-derived, but not CNS-resident, macrophages. These findings provide the first evidence of trace aminergic and purinergic crosstalk, and support the potential for TAAR1 as a novel therapeutic target in inflammatory disorders.

Proteomic and Transcriptomic Profiling Reveals Insights into Mechanisms of Leukemic Immune Escape

Despite the high rates of complete remissions achievable by modern frontline therapies, relapse of acute lymphoblastic leukemia (ALL) remains common and is associated with poor long-term survival. Relapse occurs due to the outgrowth of cells from the residual leukemia niche. These are able to evade populations of leukemia-specific memory/effector CD8+ and CD4+ T-cells that are robustly detectable in most patients. This implies a need for better understanding of basic mechanisms underlying relapse and leukemic immune evasion. The presence of phenotypically exhausted PD1+/- TIM3+ CD4+ T-cells in diagnostic bone marrow biopsy samples has been associated with inferior clinical outcomes in clinical studies of ALL patients. This suggests that CD4+ T-cell dysfunction facilitates immune escape by residual leukemic cells. To examine this, we used single-cell RNAseq/CITESEq to study CD4+ T-cells in diagnostic bone marrow biopsy samples and a murine model of BCR-ABL-rearranged ALL. In both settings, we discovered that FOXP3-PD1+ TIM3+ CD4+ T-cells are primarily composed of a unique population capable of helper and cytotoxic functions ("T-helper cytotoxic" or THCTX cells). THCTX cells expressed the effector cytokines TNF and IFNγ, but also the suppressive cytokine IL-10. In advanced leukemia, THCTX cells maintained IFNγ expression but lost the capacity for TNF synthesis, suggestive of a unique state of exhaustion. Administration of an anti-PDL1 antibody in combination with the BCR-ABL inhibitor nilotinib markedly improved long-term survival from 10% to 70% in preclinical models. Treatment benefit from dual therapy was dependent on the presence of CD4+ T-cells and was associated with the clonal expansion of leukemia-specific THCTX cells. Expanded THCTX clones had relatively preserved expression of the helper molecule CD40L as well as of ligands for the CCR5 chemokine receptor, which plays a key role in recruitment of memory/effector T-cells. Congruent with these observations, anti-PDL1 checkpoint blockade was also associated with higher frequencies of Granzyme+ CD8+ T-cells. T-cell exhaustion occurs due to chronic TCR stimulation occurring in the presence of concerted microenvironmental cues from surrounding cell types. To understand how the topography of the leukemia niche contributes to the observed exhaustion of CD4+ T-cells, we performed spatial proteomic profiling of mouse splenic tissue during leukemia development. Leukemic cells express GFP and were recognized with an anti-GFP antibody. Early leukemic infiltration led to pronounced disruption of the immune structures of the spleen and the adoption of novel cell neighborhoods (Figure 1). Infiltration was marked by loss of boundaries between the red and white pulp, and dissolution of the marginal and mantle zones. CD4+ and CD8+ T-cells were diminished in frequency, infrequently observed in proximity to leukemic cells, and expressed exhaustion markers, suggestive of an immune desert phenotype. Overall, these findings support a model in which ALL progression occurs due to the accrued dysfunction of CD4+ THCTX cells marked by a loss of helper and chemotactic functions. This is coincident with profound disruption and reorganization of the lymphoid environment into T-cell poor zones. These findings provide mechanistic insights into the high relapse risk observed among patients with CD4+ T-cell phenotypic exhaustion. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Participation of MAPK and PI3K in Regulation of Cytokine Secretion by Peripheral Blood Monocular Cells in Response to Escherichia coli LPS and rDer p 2 Combination.

Search for the effective approaches to treat acute inflammation caused by combination of allergens and infectious agents is an important task for public health worldwide. House dust mites Dermatophagoides pteronyssinus are the source of allergens of the Der p groups and of microbial compounds, in particular, lipopolysaccharides (LPS). LPS and Der p 2 induce secretion of pro-inflammatory cytokines via activation of kinases p38 MAPK, MEK1/2, and PI3K. Participation of these kinases in the regulation of cells response to combined exposure to LPS and Der p 2 has not been sufficiently studied. We studied the effects of kinases (p38 MAPK, MEK1/2, and PI3K) inhibition on secretion of cytokines (TNF, IL-8, and IL-6) by peripheral blood mononuclear cells (PBMC) of healthy volunteers in response to E. coli LPS and rDer p 2. Contribution of kinases to the regulation of cell response to different agents (rDer p 2 and/or LPS) was revealed. It was found that p38 MAPK plays a key role in the regulation of secretion TNF by PBMC in response to the combination of LPS and rDer p 2. MEK1/2-dependent signaling is the main pathway for the synthesis of TNF and IL-8 in response to LPS and rDer p 2. PI3K-dependent signaling negatively regulates TNF production during rDer p 2-induced cell activation, but is not involved in the response to the combination of LPS and rDer p 2. PI3K-dependent signaling in the regulation of PBMC cytokine synthesis is most pronounced in response to their activation by rDer p 2. Understanding the mechanisms of immune cell responses to combinations of inflammatory agents could facilitate the search for new intracellular targets for anti-inflammatory therapy.

Identification of a Distal Locus Enhancer Element That Controls Cell Type-Specific TNF and LTA Gene Expression in Human T Cells.

The human TNF/LT locus genes TNF, LTA, and LTB are expressed in a cell type-specific manner. In this study, we show that a highly conserved NFAT binding site within the distal noncoding element hHS-8 coordinately controls TNF and LTA gene expression in human T cells. Upon activation of primary human CD4+ T cells, hHS-8 and the TNF and LTA promoters display increased H3K27 acetylation and nuclease sensitivity and coordinate induction of TNF, LTA, and hHS-8 enhancer RNA transcription occurs. Functional analyses using CRISPR/dead(d)Cas9 targeting of the hHS-8-NFAT site in the human T cell line CEM demonstrate significant reduction of TNF and LTA mRNA synthesis and of RNA polymerase II recruitment to their promoters. These studies elucidate how a distal element regulates the inducible cell type-specific gene expression program of the human TNF/LT locus and provide an approach for modulation of TNF and LTA transcription in human disease using CRISPR/dCas9.

CX3CL1 and IL-15 Promote CD8 T cell chemoattraction in HIV and in atherosclerosis.

Atherosclerotic cardiovascular disease (ASCVD) remains an important cause of morbidity in the general population and risk for ASCVD is increased approximately 2-fold in persons living with HIV infection (PLWH). This risk is linked to elevated CD8 T cell counts that are abundant in atherosclerotic plaques and have been implicated in disease pathogenesis yet the mechanisms driving T cell recruitment to and activation within plaques are poorly defined. Here we investigated the role of CD8 T cells in atherosclerosis in a non-human primate model of HIV infection and in the HIV-uninfected elderly; we sought to identify factors that promote the activation, function, and recruitment to endothelium of CX3CR1+ CD8 T cells. We measured elevated expression of CX3CL1 and IL-15, and increased CD8 T cell numbers in the aortas of rhesus macaques infected with SIV or SHIV, and demonstrated similar findings in atherosclerotic vessels of HIV-uninfected humans. We found that recombinant TNF enhanced the production and release of CX3CL1 and bioactive IL-15 from aortic endothelial cells, but not from aortic smooth muscle cells. IL-15 in turn promoted CX3CR1 surface expression on and TNF synthesis by CD8 T cells, and IL-15-treated CD8 T cells exhibited enhanced CX3CL1-dependent chemoattraction toward endothelial cells in vitro. Finally, we show that CD8 T cells in human atherosclerotic plaques have an activated, resident phenotype consistent with in vivo IL-15 and CX3CL1 exposure. In this report, we define a novel model of CD8 T cell involvement in atherosclerosis whereby CX3CL1 and IL-15 operate in tandem within the vascular endothelium to promote infiltration by activated CX3CR1+ memory CD8 T cells that drive further endothelial activation via TNF. We propose that these interactions are prevalent in aging and in PLWH, populations where circulating activated CX3CR1+ CD8 T cell numbers are often expanded.

Inhibitors of Tumor Necrosis Factor Synthesis as a New Approach for theTreatment of Rheumatoid Arthritis

Objective: The treatment of rheumatoid arthritis (RA) is based on the inhibition of TNF. Here we evaluated whether drugs that might inhibit TNF, such as pentoxifylline (PTX), rupatadine (RUP), rolipram (ROL) and thalidomide (THA), could be an alternative for RA treatment. Methods: In wistar male rats the changes in paw thickness, plasma TNF and by the activity of basic aminopeptidase (APB) in soluble fraction of synovial tissue and peripheral blood mononuclear cells (PBMC) evaluated after daily injection for 30 days were taken as anti-inflammatory outputs, while hepatotoxicity was assessed by measuring plasma alanine transaminase (ALT) and aspartate transaminase (AST) activity. The content of IL1-?, IL-6 in serum and synovial fluid and the histology of the injured tissue were determined only for ROL, THA and ROL+THA. Prednisolone was used as a standard drug. Results: Collagen treatment induced paw thickness, histological changes in the tibiotarsal joint, increase in synovial fluid of both cytokines and synovial tissue of APB activity. Furthermore, the APB activity in PBMC was reduced and ALT and AST activity were enhanced. The most effective drug schedule in reducing arthritis induced changes described above, as well as recovering from control levels TNF, IL1-β, APB in synovial tissue and AST activities were THA and the association of ROL and THA. However, only THA alone reduced the levels of ALT. Conclusion: The synthesis of TNF in RA models can be blocked by drugs acting at different targets. We show that THA and THA+ROL emerges as simple and effective therapeutic alternatives for RA.

The specific ex vivo released cytokine profile is associated with ischemic stroke outcome and improves its prediction

BackgroundInflammation is associated with poor outcome after stroke. A relationship between ex vivo cytokine synthesis and stroke outcome remains unclear. We explored an association between ex vivo cytokine release, circulating interleukin (IL)-6 as a marker of systemic inflammation, and stroke prognosis. We assessed the utility of ex vivo synthesized cytokines for predicting stroke outcome.MethodsWe collected blood from 248 ischemic stroke patients and stimulated it ex vivo with lipopolysaccharide. We measured concentration of synthesized cytokines (TNFα, IP-10, IL-1β, IL-6, IL-8, IL-10, and IL-12) and plasma IL-6. We assessed functional outcome 3 months after stroke using the modified Rankin Scale. To assess the prognostic ability of cytokines, we applied multivariate logistic regression, cluster analysis, and construction of multimarker score.ResultsDecreased release of IP-10, TNFα, IL-1β, and IL-12; increased release of IL-10 and IL-8; and higher plasma IL-6 level were associated with poor outcome. Cluster analysis identified three groups of patients with distinct cytokine profiles. The group with the worst outcome demonstrated high synthesis of IL-10, IL-8, IL-1β, and IL-6 and low synthesis of IL-12, IP-10, and TNFα accompanied by high circulating IL-6 level. The group with the best prognosis showed high synthesis of TNFα, IP-10, IL-12, IL-1β, and IL-6; low synthesis of IL-10 and IL-8; and low plasma IL-6. Patients with intermediate outcome had low synthesis of all cytokines accompanied by low circulating IL-6. We constructed a multimarker score composed of ex vivo released IL-12, IL-10, TNFα, and plasma IL-6. Addition of this score to clinical variables led to significant increase in c-statistic (0.81 vs 0.73, p = 0.02) and net reclassification improvement.ConclusionThe decreased ex vivo release of pro-inflammatory cytokines and increased release of IL-10 and IL-8 are related to poor outcome after stroke. Cytokine-based multimarker score adds prognostic value to clinical model for predicting stroke outcome.

Mast Cell Degranulation Exacerbates Skin Rejection by Enhancing Neutrophil Recruitment.

Recent evidences indicate an important role of tissue inflammatory responses by innate immune cells in allograft acceptance and survival. Here we investigated the role of mast cells (MC) in an acute male to female skin allograft rejection model using red MC and basophil (RMB) mice enabling conditional MC depletion. Kinetic analysis showed that MCs markedly accelerate skin rejection. They induced an early inflammatory response through degranulation and boosted local synthesis of KC, MIP-2, and TNF. This enhanced early neutrophil infiltration compared to a female-female graft-associated repair response. The uncontrolled neutrophil influx accelerated rejection as antibody-mediated depletion of neutrophils delayed skin rejection. Administration of cromolyn, a MC stabilizer and to a lesser extent ketotifen, a histamine type I receptor antagonist, and absence of MCPT4 chymase also delayed graft rejection. Together our data indicate that mediators contained in secretory granules of MC promote an inflammatory response with enhanced neutrophil infiltration that accelerate graft rejection.

Benefits of VCE-003.2, a cannabigerol quinone derivative, against inflammation-driven neuronal deterioration in experimental Parkinson\u2019s disease: possible involvement of different binding sites at the PPAR\u03b3 receptor

BackgroundNeuroprotection with cannabinoids in Parkinson’s disease (PD) has been afforded predominantly with antioxidant or anti-inflammatory cannabinoids. In the present study, we investigated the anti-inflammatory and neuroprotective properties of VCE-003.2, a quinone derivative of the non-psychotrophic phytocannabinoid cannabigerol (CBG), which may derive its activity at the peroxisome proliferator-activated receptor-γ (PPARγ). The compound is also an antioxidant.MethodsWe evaluated VCE-003.2 in an in vivo [mice subjected to unilateral intrastriatal injections of lipopolysaccharide (LPS)] model of PD, as well as in in vitro (LPS-exposed BV2 cells and M-213 cells treated with conditioned media generated from LPS-exposed BV2 cells) cellular models. The type of interaction of VCE-003.2 at the PPARγ receptor was furtherly investigated in bone marrow-derived human mesenchymal stem cells (MSCs) and sustained with transcriptional assays and in silico docking studies.ResultsVCE-003.2 has no activity at the cannabinoid receptors, a fact that we confirmed in this study using competition studies. The administration of VCE-003.2 to LPS-lesioned mice attenuated the loss of tyrosine hydroxylase (TH)-containing nigrostriatal neurons and, in particular, the intense microgliosis provoked by LPS in the substantia nigra, measured by Iba-1/Cd68 immunostaining. The analysis by qPCR of proinflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and inducible nitric oxide synthase (iNOS) in the striatum showed they were markedly elevated by the LPS lesion and strongly reduced by the treatment with VCE-003.2. The effects of VCE-003.2 in LPS-lesioned mice implied the activation of PPARγ receptors, as they were attenuated when VCE-003.2 was co-administered with the PPARγ inhibitor T0070907. We then moved to some in vitro approaches, first to confirm the anti-inflammatory profile of VCE-003.2 in cultured BV2 cells exposed to LPS. VCE-003.2 was able to attenuate the synthesis and release of TNF-α and IL-1β, as well as the induction of iNOS and cyclooxygenase-2 (COX-2) elicited by LPS in these cells. However, we found such effects were not reversed by GW9662, another classic PPARγ antagonist. Next, we investigated the neuroprotective effects of VCE-003.2 in cultured M-213 neuronal cells exposed to conditioned media generated from LPS-exposed cultured BV2 cells. VCE-003.2 reduced M-213 cell death, but again, such effects were not reversed by T0070907. Using docking analysis, we detected that VCE-003.2 binds both the canonical and the alternative binding sites in the PPARγ ligand-binding pocket (LBP). Functional assays further showed that T0070907 almost abolished PPARγ transcriptional activity induced by rosiglitazone (RGZ), but it did not affect the activity of VCE-003.2 in a Gal4-Luc system. However, T0070907 inhibited the effects of RGZ and VCE-003.2 on the expression of PPARγ-dependent genes upregulated in MSCs.ConclusionsWe have demonstrated that VCE-003.2 is neuroprotective against inflammation-driven neuronal damage in an in vivo model of PD and in in vitro cellular models of neuroinflammation. Such effects might involve PPARγ receptors, although in silico and in vitro experiments strongly suggest that VCE-003.2 targets PPARγ by acting through two binding sites at the LBP, one that is sensitive to T0070907 (canonical binding site) and other that is not affected by this PPARγ antagonist (alternative binding site).

NF-κB regulates GDF-15 to suppress macrophage surveillance during early tumor development.

Macrophages are attracted to developing tumors and can participate in immune surveillance to eliminate neoplastic cells. In response, neoplastic cells utilize NF-κB to suppress this killing activity, but the mechanisms underlying their self-protection remain unclear. Here, we report that this dynamic interaction between tumor cells and macrophages is integrally linked by a soluble factor identified as growth and differentiation factor 15 (GDF-15). In vitro, tumor-derived GDF-15 signals in macrophages to suppress their proapoptotic activity by inhibiting TNF and nitric oxide (NO) production. In vivo, depletion of GDF-15 in Ras-driven tumor xenografts and in an orthotopic model of pancreatic cancer delayed tumor development. This delay correlated with increased infiltrating antitumor macrophages. Further, production of GDF-15 is directly regulated by NF-κB, and the colocalization of activated NF-κB and GDF-15 in epithelial ducts of human pancreatic adenocarcinoma supports the importance of this observation. Mechanistically, we found that GDF-15 suppresses macrophage activity by inhibiting TGF-β-activated kinase (TAK1) signaling to NF-κB, thereby blocking synthesis of TNF and NO. Based on these results, we propose that the NF-κB/GDF-15 regulatory axis is important for tumor cells in evading macrophage immune surveillance during the early stages of tumorigenesis.

Stimulation of nAchRα7 Receptor Inhibits TNF Synthesis and Secretion in Response to LPS Treatment of Mast Cells by Targeting ERK1/2 and TACE Activation.

The cholinergic anti-inflammatory pathway is recognized as one of the main mechanisms of neuromodulation of the immune system. Activation of the α7 nicotinic acetylcholine receptor (nAchRα7) suppresses cytokine synthesis in distinct immune cells but the molecular mechanisms behind this effect remain to be fully described. Mast cells (MCs) are essential players of allergic reactions and innate immunity responses related to chronic inflammation. Activation of TLR4 receptor in MCs leads to the rapid secretion of pre-synthesized TNF from intracellular pools and to the activation of NFκB, necessary for de novo synthesis of TNF and other cytokines. Here we report that the nAchRα7 receptor specific agonist GTS-21 inhibits TLR4-induced secretion of preformed TNF from MCs in vivo and in vitro. Utilizing bone marrow-derived mast cells (BMMCs) it was found that GTS-21 also diminished secretion of de novo synthesized TNF, TNF mRNA accumulation and IKK-dependent p65-NFκB phosphorylation in response to LPS. nAchRα7 triggering prevented TLR4-induced ERK1/2 phosphorylation, which resulted an essential step for TNF secretion due to the phosphorylation of the metallopeptidase responsible for TNF maturation (TACE). Main inhibitory actions of GTS-21 were prevented by AG490, an inhibitor of JAK-2 kinase. Our results show for the first time, that besides the prevention of NFκB-dependent transcription, inhibitory actions of nAchRα7 triggering include the blockade of pathways leading to exocytosis of granule-stored cytokines in MCs.

Persistent p55TNFR expression impairs T cell responses during chronic tuberculosis and promotes reactivation.

The pleiotropic activities of TNF are mediated by two structurally related but functionally distinct type I transmembrane receptors, p55TNFR and p75TNFR expressed in most cell types, that can be cleaved and act as TNF scavengers. Here, we investigated the effect of persistent p55TNFR cell surface expression during aerosol inhalation challenge with virulent M. tuberculosis H37Rv. We demonstrated that persistency of p55TNFR in macrophage cultures increased the synthesis of soluble TNF, p75TNFR and NO, however, had no effects on bacteria killing ability. Furthermore, it did not facilitate enhanced protection to primary acute M. tuberculosis infection in p55∆NS mice. Without exacerbated lung inflammation, we found a compensatory increase in p75TNFR shedding and decrease in bioactive TNF in BAL of p55∆NS mice after M. tuberculosis challenge. Defective expressions of CD44 and INFγ attributed to an impaired T cell response during persistent p55TNFR expression that caused marginal transient susceptibility during chronic infection. Moreover, persistent p55TNFR expression induced early reactivation during latent tuberculosis infection. These data indicate a prominent role of p55TNFR shedding in Th1 mediated protection against chronic and latent tuberculosis infection.

Anti-TNF Therapy.

Tumor necrosis factor (TNF) is one of the most important cytokines produced by macrophages. TNF is a very important component of host defense, released very rapidly after all types of injuries and stimuli. The kinetics of TNF release are short, and so it is perhaps not surprising that prolonged TNF production is associated with pathology. This was first elucidated in rheumatoid arthritis but extends to other chronic inflammatory diseases such as Crohn's disease and psoriasis. In this chapter, the discovery of anti-TNF therapy is reviewed, with its benefit but also its limitations. The potential of anti-TNF therapy in other diseases, e.g., cardiovascular and fibrosis, is discussed, as is the opportunity to define ways of blocking TNF synthesis.

Trace Levels of Staphylococcal Enterotoxin Bioactivity Are Concealed in a Mucosal Niche during Pulmonary Inflammation.

Pathogen and cellular by-products released during infection or trauma are critical for initiating mucosal inflammation. The localization of these factors, their bioactivity and natural countermeasures remain unclear. This concept was studied in mice undergoing pulmonary inflammation after Staphylococcal enterotoxin A (SEA) inhalation. Highly purified bronchoalveolar lavage fluid (BALF) fractions obtained by sequential chromatography were screened for bioactivity and subjected to mass spectrometry. The Inflammatory and inhibitory potentials of the identified proteins were measured using T cells assays. A potent pro-inflammatory factor was detected in BALF, and we hypothesized SEA could be recovered with its biological activity. Highly purified BALF fractions with bioactivity were subjected to mass spectrometry. SEA was the only identified protein with known inflammatory potential, and unexpectedly, it co-purified with immunosuppressive proteins. Among them was lactoferrin, which inhibited SEA and anti-CD3/-CD28 stimulation by promoting T cell death and reducing TNF synthesis. Higher doses of lactoferrin were required to inhibit effector compared to resting T cells. Inhibition relied on the continual presence of lactoferrin rather than a programming event. The data show a fraction of bioactive SEA resided in a mucosal niche within BALF even after the initiation of inflammation. These results may have clinical value in human diagnostic since traces levels of SEA can be detected using a sensitive bioassay, and may help pinpoint potential mediators of lung inflammation when molecular approaches fail.

Trace levels of staphylococcal enterotoxin bioactivity are concealed in a mucosal niche during pulmonary inflammation (MUC1P.917)

Abstract Pathogen and cellular by-products released during infection or trauma are critical for initiating mucosal inflammation. This was studied in mice undergoing pulmonary inflammation after Staphylococcal enterotoxin A (SEA) inhalation. A potent pro-inflammatory factor was detected using a sensitive bioassay suggesting the presence of an alarmin, a danger-associated molecular pattern (DAMP), and/or a cytokine. Highly purified bronchoalveolar lavage fluid (BALF) fractions obtained by sequential chromatography were screened for bioactivity and subjected to mass spectrometry. SEA was the only identified protein with known inflammatory potential, and unexpectedly, it co-purified with immunosuppressive proteins. Among them was lactoferrin, which inhibited SEA and anti-CD3/-CD28 stimulation by promoting T cell death and reducing TNF synthesis. Higher doses of lactoferrin were required to inhibit effector compared to resting T cells. This inhibition relied on the continual presence of lactoferrin rather than a programming event. The data show that bioactive SEA is constituted in a mucosal niche within BALF even after the initiation of inflammation. These results may have clinical value in human diagnostic settings since traces levels of SEA can be detected using a sensitive bioassay, and may help pinpoint potential mediators of lung inflammation when molecular approaches fail. Submitted to American Journal of Respiratory Cell and Molecular Biology.

Cathelicidin rCRAMP stimulates rat mast cells to generate cysteinyl leukotrienes, synthesize TNF and migrate: involvement of PLC/A2, PI3K and MAPK signaling pathways.

Cathelicidins represent a family of cationic peptides involved in host defense systems. Apart from exerting direct anti-microbial effects, cathelicidins can regulate immune responses by affecting the activity of cells playing a role in antibacterial defense. Taking into account that mast cells are critical components of host defense, the aim of this study was to determine whether rat cathelicidin-related anti-microbial peptide (rCRAMP) can influence mast cell activity. We have demonstrated that activation of fully mature rat mast cells with rCRAMP resulted in generation and release of cysteinyl leukotrienes (cysLTs). However, rCRAMP failed to induce mast cell degranulation and histamine release. We also found that rCRAMP stimulated rat mast cells to synthesize TNF, but not CXCL8. What is more, this peptide induced GM-CSF, IL-1β, CCL2 and CCL3 but not IL-33 mRNA expression in mast cells. Finally, we showed that this cathelicidin serves as potent chemoattractant for rat mast cells. rCRAMP-mediated cysLT synthesis and mast cell migration were strongly inhibited by IL-10 pre-treatment. With the use of specific inhibitors, we established that activation of PLC/A2 and ERK1/2, but not p38, was required for rCRAMP-induced mast cell stimulation, while PI3K-dependent pathway is involved in both TNF synthesis and mast cell migration. Our results suggest that cathelicidins can amplify inflammatory responses by causing mast cells accumulation and by stimulating these cells to release potent pro-inflammatory mediators.

AB0101 Articular Adipose Tissue from Osteoarthritis Patients is A Source of Cytokines and Adipokines

BackgroundRecently rheumatoid adipose tissue has been suggested to play an important role in chronic inflammatory joint diseases. Its ability to synthesize cytokines and adipokines has been characterized also in our...

Haplotype Analysis of TNFA Gene in Peptic Ulcer Patients

The TNFA gene product TNF- is a cytokine promoting an immune response after bacterial infections. An excessive production of the cytokine is thought to be connected with Helicobacter pylori-induced disorders like gastritis, peptic ulcer disease (PUD), intestinal metaplasia, or even gastric cancer. The synthesis of TNF-a is controlled at transcriptional level and dependent on single nucleotide polymorphisms (SNPs) of TNFA promoter region. SNPs assembled in haplotype have been implicated as potential risk factors for various autoimmune and infectious diseases. The aim of this study was to determine the frequencies of TNFA haplotypes composed of the four common single-nucleotide polymorphisms of the gene (-1031C>T: rs1799964, -863C>A: rs1800630, 857C>T: rs1799724, -308G>A: rs1800629) in peptic ulcer patients and to assess the significance of the haplotypes as the risk factors of H. pylori infection development. 203 peptic ulcer patients were genotyped using polymerase chain reaction-restriction fragment length polymorphism method. Haplotypes and degree of linkage disequilibrium (LD) were inferred with PHASE 2.1 and EMLD software. There was no statistically significant difference in haplotype frequencies between the H. pylori-infected and -uninfected peptic ulcer cases (p=0.62). Analogous association was also absent in the subgroups: peptic ulcer woman (p=0.69), peptic ulcer men (p=0.17). The locus pairs -308_-857, -863_-1031, and -857_-1031 was found to be in very strong LD , -308_-1031 and -857 and -863 in strong LD, and -308_-863 in modest LD. TNFA haplotype structure is not connected with individual differences in susceptibility to development of H. pylori infection in peptic ulcer patients. Address for correspondence: Dr. Aleksandra Salagacka Medical University of Lodz Musuynskiego 1, 90-151, Lodz, Poland Telephone/Fax: +48 42 677 91 30 E-mail: aleksandra.salagacka@umed.lodz.pl INTRODUCTION Tumour necrosis factor-alpha (TNF-) is a cytokine known for having the multitude of functions. One of the most prominent is to promote of immune response after bacterial infections. In gastric mucosa infected by Helicobacter pylori TNF- prompts recruitment of mononuclear cells and neutrophils into the gastric lamina propria (Crabtree 1991; Huseyinov 1999). An excessive production of the cytokine could also exert some pathogenic effects (Beutler 1993) and is thought to be connected with H. pylori-induced disorders like gastritis, peptic ulcer disease (PUD), intestinal metaplasia, or even gastric cancer (Goto 2003; Shanks 2009). The synthesis of TNF- is controlled at transcriptional level in a cell typeand stimulus-specific manner involving different transcription factors to a promoter region of the TNFA encoding TNF- (Falvo 2010). An important role in the regulation of TNFA expression is attributed to single nucleotide polymorphisms (SNPs) of promoter region of the gene. Accordingly, these genetic variations are implicated as potential risk factors for various autoimmune and infectious diseases. To date, SNPs at positions -238, -308, -857, 863, -1031 of TNFA have been investigated in various populations afflicted by PUD or other H. pylori-induced disorders (Lee 2005; Lu 2005; Lee 2004; Wilschanski 2007; Kim 2006; Machado 2003). Unfortunately, studies focused on determining the importance of individual TNFA loci have produced non-significant or conflicting results. However, some evidence has supported the hypothesis that effects exerts by these SNPs could be observed or are more pronounced when the polymorphisms are researched and analysed concomitantly. Studies by Lu et al. (2005) conducted in Taiwanese revealed that risk of ulceration after H. pylori infection was higher for carriers of both -1031C or -863A allele of TNFA than for individuals having only one of the mentioned alleles. Similarly, the risk of gastric ulcers among Japanese was found to be greatest in simultaneous carriers of so called ‘high-producer’ alleles of TNFA -857/-863/-1031 than when the presence of these particular alleles was stated individually (Sugimoto 2007). 10 ALEKSANDRA SALAGACKA, MARTA ZEBROWSKA, AGNIESZKA JELEN ET AL Some research results could imply the best approach to investigate the relevancy of TNFA variations, in the development of gastroduodenal diseases, is to explore haplotype structure of TNFA gene. Although Chakravorty et al. (2008) did not stated any connection between single TNFA loci (-308, -857, -863, -1031) and H. pylorimediated duodenal ulcer, the researchers found the TNFA haplotype -308G_-857C_-863A_-1031T in H. pylori-infected duodenal ulcer individuals was significantly more frequent than in the infected but ulcer-free cases. Thus far, haplotype analysis of the gene has been applied in the studies on type 1 diabetes (Stayoussef 2010), gastric cancer (Canedo 2008), iron deficiency anemia (Atkinson 2008), Behcet’s disease (Park 2006), schizophrenia (Saviouk 2005), juvenile oligoarthritis (Zeggini 2002), hepatitis C virus infection (Thio 2004), and some others. Recently, the researchers investigated association between individual -308G>A or -1031T>C SNPs of TNFA and peptic ulcer risk in Poles (Salagacka 2014). In this study the researchers conducted haplotype analysis of TNFA combining mentioned genotyping data and newly obtained for -308 locus and two further -857 and -863 loci of TNFA. The aim of presented research was to estimate the TNFA haplotype structure and its significance in development peptic ulcer disease in Polish population.