Levels Of Pro-inflammatory Cytokine Production Research Articles

Hyperexpression of Proinflammatory Cytokines in Blood as a Biomarker of Systemic Inflammatory Response in Schizophrenia: Scoping Review

Introduction: An imbalance of the genetically determined cytokine response plays a key role in the etiology of treatment-resistant schizophrenia (TRS). In recent years, an attempt has been made to evaluate the prognostic role of systemic inflammation in the development of TRS. The problem requires a multidisciplinary approach on the part of the specialists in the following clinical disciplines: psychiatry, immunology, experimental medicine and pharmacogenetics. The solution of this problem is possible with the involvement of preventive and personalized medicine. The purpose: Evaluation the prognostic role of genetic polymorphisms of pro-inflammatory cytokines in the development of TRS. Materials and Methods: We conducted a keyword-based analysis of the English and Russian-language articles published within the past 5 years. The following databases were used in the study: PubMed, MedLine, Web of Science Core Collection (Clarivate Analytics), Web Science, Russian Science Citation Index, Scopus, Scientific Research, Google Scholar, Oxford Press, and eLibrary. Results: In a number of the analyzed works, an increased level of pro-inflammatory cytokine production was noted in patients with TRS. Based on this, single nucleotide variants (SNVs), their influence on the expression of pro- and anti-inflammatory cytokine genes, as well as their predictor role in the development of TRS. The most promising SNVs for further studies were identified. Conclusion: The risk of developing TRS is associated with a genetically determined status of the cytokine response and its regulation. Studies of the association of various SNVs of genes encoding pro-inflammatory cytokines in the Russian Federation need to be continued.

Macrophage β2-AR activation amplifies inflammation in wound healing by upregulating Trem1 via the cAMP/PKA/CREB pathway

BackgroundInflammation is an important part of the wound healing process. The stress hormone epinephrine has been demonstrated to modulate the inflammatory response via its interaction with β2-adrenergic receptor (β2-AR). However, the precise molecular mechanism through which β2-AR exerts its influence on inflammation during the wound healing process remains an unresolved question. MethodsTranscriptome datasets of wound and macrophages from the GEO database were reanalyzed using bioinformatics. The role of β2-AR in wound healing was explored by a mouse hind paw plantar wound model, and histological analyses were performed to assess wound healing. In vivo and in vitro assays were performed to elucidate the role of β2-AR on the inflammatory response. Triggering receptor expressed on myeloid cells 1 (Trem1) was knocked down with siRNA on RAW cells and western blot and qPCR assays were performed. ResultsTrem1 was upregulated within 24 h of wounding, and macrophage β2-AR activation also upregulated Trem1. In vivo experiments demonstrated that β2-AR agonists impaired wound healing, accompanied by upregulation of Trem1 and activation of cAMP/PKA/CREB pathway, as well as by a high level of pro-inflammatory cytokine production. In vitro experiments showed that macrophage β2-AR activation amplified LPS-induced inflammation, and knockdown of Trem1 reversed this effect. Using activator and inhibitor of cAMP, macrophage β2-AR activation was confirmed to upregulate Trem1 via the cAMP/PKA/CREB pathway. ConclusionOur study found that β2-AR agonists increase Trem1 expression in wounds, accompanied by amplification of the inflammatory response, impairing wound healing. β2-AR activation in RAW cells induces Trem1 upregulation via the cAMP/PKA/CREB pathway and amplifies LPS-induced inflammatory responses.

Photodynamic anti-inflammatory activity of meso‑aryl substituted porphyrin derivative on mammalian macrophages

BackgroundOur group focused on a meso‑aryl substituted porphyrin molecule for its photodynamic anti-inflammatory activities on the mammalian macrophages. Materials and methodsThe porphyrine derivative previously synthesized in this study was synthesized and characterized by 1H NMR. We then examined their immunomodulatory activities based on the changes in the pro-inflammatory cytokine production levels after LPS stimulation in dark and light activated conditions. ResultsOur results suggest that porphyrin derivative had anti-inflammatory photodynamic activity in vitro at subtoxic concentrations. Our study aims to pave a way for anti-inflammatory photodynamic therapy application in the inflammatory and autoimmune disorders. Most of the studies either focus on photodynamic cytotoxicity of the porphyrin derivatives to suppress the inflammation or porphyrin derivatives’ anti-inflammatory activity without the photodynamic activation. ConclusionOur future studies will focus on the generation and in vitro as well as in vivo characterization of the porphyrin derivatives with anti-inflammatory photodynamic therapy applications. In this way, novel drug candidates that would have lower side effects can be generated for the patients.

CLEC5A expression can be triggered by spike glycoprotein and may be a potential target for COVID-19 therapy.

The immune response is crucial for coronavirus disease 19 (COVID-19) progression, with the participation of proinflammatory cells and cytokines, inducing lung injury and loss of respiratory function. CLEC5A expression on monocytes can be triggered by viral and bacterial infections, leading to poor outcomes. Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) is able to induce neutrophil activation by CLEC5A and Toll-like receptor 2, leading to an aggressive inflammatory cascade, but little is known about the molecular interactions between CLEC5A and SARS-CoV-2 proteins. Here, we aimed to explore how CLEC5A expression could be affected by SARS-CoV-2 infection using immunological tools with in vitro, in vivo, and in silico assays. The findings revealed that high levels of CLEC5A expression were found in monocytes from severe COVID-19 patients in comparison with mild COVID-19 and unexposed subjects, but not in vaccinated subjects who developed mild COVID-19. In hamsters, we detected CLEC5A gene expression during 3-15 days of Omicron strain viral challenge. Our results also showed that CLEC5A can interact with SARS-CoV-2, promoting inflammatory cytokine production, probably through an interaction with the receptor-binding domain in the N-acetylglucosamine binding site (NAG-601). The high expression of CLEC5A and high levels of proinflammatory cytokine production were reduced in vitro by a human CLEC5A monoclonal antibody. Finally, CLEC5A was triggered by spike glycoprotein, suggesting its involvement in COVID-19 progression; therapy with a monoclonal antibody could be a good strategy for COVID-19 treatment, but vaccines are still the best option to avoid hospitalization/deaths.

SARS-CoV-2 host-shutoff impacts innate NK cell functions, but antibody-dependent NK activity is strongly activated through non-spike antibodies.

The outcome of infection is dependent on the ability of viruses to manipulate the infected cell to evade immunity, and the ability of the immune response to overcome this evasion. Understanding this process is key to understanding pathogenesis, genetic risk factors, and both natural and vaccine-induced immunity. SARS-CoV-2 antagonises the innate interferon response, but whether it manipulates innate cellular immunity is unclear. An unbiased proteomic analysis determined how cell surface protein expression is altered on SARS-CoV-2-infected lung epithelial cells, showing downregulation of activating NK ligands B7-H6, MICA, ULBP2, and Nectin1, with minimal effects on MHC-I. This occurred at the level of protein synthesis, could be mediated by Nsp1 and Nsp14, and correlated with a reduction in NK cell activation. This identifies a novel mechanism by which SARS-CoV-2 host-shutoff antagonises innate immunity. Later in the disease process, strong antibody-dependent NK cell activation (ADNKA) developed. These responses were sustained for at least 6 months in most patients, and led to high levels of pro-inflammatory cytokine production. Depletion of spike-specific antibodies confirmed their dominant role in neutralisation, but these antibodies played only a minor role in ADNKA compared to antibodies to other proteins, including ORF3a, Membrane, and Nucleocapsid. In contrast, ADNKA induced following vaccination was focussed solely on spike, was weaker than ADNKA following natural infection, and was not boosted by the second dose. These insights have important implications for understanding disease progression, vaccine efficacy, and vaccine design.

Intracellular NAD+ Depletion Confers a Priming Signal for NLRP3 Inflammasome Activation.

Nicotinamide adenine dinucleotide (NAD+) is an important cofactor in many redox and non-redox NAD+-consuming enzyme reactions. Intracellular NAD+ level steadily declines with age, but its role in the innate immune potential of myeloid cells remains elusive. In this study, we explored whether NAD+ depletion by FK866, a highly specific inhibitor of the NAD salvage pathway, can affect pattern recognition receptor-mediated responses in macrophages. NAD+-depleted mouse bone marrow-derived macrophages (BMDMs) exhibited similar levels of proinflammatory cytokine production in response to LPS or poly (I:C) stimulation compared with untreated cells. Instead, FK866 facilitated robust caspase-1 activation in BMDMs in the presence of NLRP3-activating signals such as ATP and nigericin, a potassium ionophore. However, this FK866-mediated caspase-1 activation was completely abolished in Nlrp3-deficient macrophages. FK866 plus nigericin stimulation caused an NLRP3-dependent assembly of inflammasome complex. In contrast, restoration of NAD+ level by supplementation with nicotinamide mononucleotide abrogated the FK866-mediated caspase-1 cleavage. FK866 did not induce or increase the expression levels of NLRP3 and interleukin (IL)-1β but drove mitochondrial retrograde transport into the perinuclear region. FK866-nigericin-induced mitochondrial transport is critical for caspase-1 cleavage in macrophages. Consistent with the in vitro experiments, intradermal coinjection of FK866 and ATP resulted in robust IL-1β expression and caspase-1 activation in the skin of wild-type, but not Nlrp3-deficient mice. Collectively, our data suggest that NAD+ depletion provides a non-transcriptional priming signal for NLRP3 activation via mitochondrial perinuclear clustering, and aging-associated NAD+ decline can trigger NLRP3 inflammasome activation in ATP-rich environments.

Novel benzoylthiourea derivatives had differential anti-inflammatory photodynamic therapy potentials on in vitro stimulated mammalian macrophages

Novel benzoylthioureas, N-((5-chloropyridin-2yl)carbamothioyl)benzamide, (HL1), N-((2-chloropyridin-3yl)carbamothioyl)benzamide, (HL2), N-((5-bromopyridin-2yl)carbamothioyl)benzamide, (HL3) and N-(Naphthalene-1-yl(phenyl)carbamothioyl)benzamide, (HL4), were synthesized. Their characterizations were made by FT-IR,1H NMR and 13C NMR spectrophotometric analysis. Single crystal X-ray diffraction measurements were conducted to determine the crystal structure of HL1 and HL4. The HL1 crystallization conditions are: in the monoclinic crystal system with P21/c space group, Z = 2, a = 8.118(2) Å, b = 12.056(3) Å, c = 13.753(4) Å. HL4crystallization conditions are: in the orthorhombic crystal system with Pbca space group, Z = 8, a = 19.597(9) Å, b = 8.270(4) Å, c = 24.299(11) Å. Investigation of photodynamic and antiinflamatory effects of these compounds revealed that they are potent adducts. Using these derivatives, mammalian macrophages were stimulated with LPS to test their anti-inflammatory activity. Based on pro-inflammatory cytokine production levels, the photodynamic anti-inflammatory activity of these adducts were found to differ. Our results showedthat benzoylthioureas can be used as potential photodynamic therapy agents to suppress the excessive inflammatory reactions encountered in autoimmune and inflammatory disorders.

HIF-1α Regulation of Cytokine Production following TLR3 Engagement in Murine Bone Marrow-Derived Macrophages Is Dependent on Viral Nucleic Acid Length and Glucose Availability.

Hypoxia-inducible factor-1α (HIF-1α) is an important regulator of glucose metabolism and inflammatory cytokine production in innate immune responses. Viruses modulate HIF-1α to support viral replication and the survival of infected cells, but it is unclear if this transcription factor also plays an important role in regulating antiviral immune responses. In this study, we found that short and long dsRNA differentially engage TLR3, inducing distinct levels of proinflammatory cytokine production (TNF-α and IL-6) in bone marrow-derived macrophages from C57BL/6 mice. These responses are associated with differential accumulation of HIF-1α, which augments NF-κB activation. Unlike TLR4 responses, increased HIF-1α following TLR3 engagement is not associated with significant alterations in glycolytic activity and was more pronounced in low glucose conditions. We also show that the mechanisms supporting HIF-1α stabilization may differ following stimulation with short versus long dsRNA and that pyruvate kinase M2 and mitochondrial reactive oxygen species play a central role in these processes. Collectively, this work suggests that HIF-1α may fine-tune proinflammatory cytokine production during early antiviral immune responses, particularly when there is limited glucose availability or under other conditions of stress. Our findings also suggest we may be able to regulate the magnitude of proinflammatory cytokine production during antiviral responses by targeting proteins or molecules that contribute to HIF-1α stabilization.

Genetic predictors of cytokine response in ENT-associated encephalitis

(1) Introduction: An imbalance of the genetically determined cytokine response plays a key role in the etiology of ENT-associated encephalitis. In recent years, an attempt has been made to evaluatethe prognostic role of chronic pathology of the paranasal sinuses in the development of acute, subacute and chronic encephalitis and meningitis, which in clinical practice are manifested both as cerebral and focal neurological symptoms and as mental disorders: from borderline to psychotic ones. The problem requires a multidisciplinary approach on the part of the specialists in the following clinical disciplines: neurology (as well as neurobiology), psychiatry, immunology, experimental medicine, otorhinolaryngology, and pharmacogenetics. The solution of this problem is possible with the involvement of preventive and personalized medicine.(2) The purpose: Evaluation the prognostic role of genetic polymorphisms of pro- and antiinflammatory cytokines in the development of ENT-associated encephalitis.(3) Materials and Methods: We conducted a keyword-based analysis of the English and Russian-language articles published within the past 30 years (from 1988 to 2018). The following databases were used in the study: PubMed, MedLine, Web of Science Core Collection (Clarivate Analytics), Web Science, Russian Science Citation Index, Scopus, Scientific Research, Google Scholar, Oxford Press, and eLibrary.(4) Results: In a number of the analyzed works, regardless of the causative agent and viral load, an increased level of pro-inflammatory cytokine production was noted in patients with more severe disease progression, neurological complications and unfavorable outcomes, both in viral encephalitis and in bacterial one. Based on this, 30 single nucleotide variants (SNV), their influence on the expression of pro- and anti-inflammatory cytokine genes, as well as their predictor role in the development of ENT-associated encephalitis were analyzed. Due to the nature of the systemic immune response, the analysis included both cerebral and extracerebral pathology-associated SNV. The inconsistency of the previously obtained results was noted, an attempt to explain this phenomenon was made. The analysis of the dynamics and geography of publications on the stated topic was made, the leading Russian scientific centers in the field were defined. The most promising SNV for further studies were identified.(5) Conclusion: The risk of developing ENT-associated encephalitis is associated with a genetically determined status of the cytokine response and its regulation. Studies of the association of various SNV of genes encoding pro- and anti-inflammatory cytokines in the Russian Federation need to be continued.

A possible beneficial effect of Bacteroides on faecal lipopolysaccharide activity and cardiovascular diseases

Faecal lipopolysaccharides (LPS) have attracted attention as potent elements to explain a correlation between the gut microbiota and cardiovascular disease (CVD) progression. However, the underlying mechanism of how specific gut bacteria contribute to faecal LPS levels remains unclear. We retrospectively analysed the data of 92 patients and found that the abundance of the genus Bacteroides was significantly and negatively correlated with faecal LPS levels. The controls showed a higher abundance of Bacteroides than that in the patients with CVD. The endotoxin units of the Bacteroides LPS, as determined by the limulus amoebocyte lysate (LAL) tests, were drastically lower than those of the Escherichia coli LPS; similarly, the Bacteroides LPS induced relatively low levels of pro-inflammatory cytokine production and did not induce sepsis in mice. Fermenting patient faecal samples in a single-batch fermentation system with Bacteroides probiotics led to a significant increase in the Bacteroides abundance, suggesting that the human gut microbiota could be manipulated toward decreasing the faecal LPS levels. In the clinical perspective, Bacteroides decrease faecal LPS levels because of their reduced LAL activity; therefore, increasing Bacteroides abundance might serve as a novel therapeutic approach to prevent CVD via reducing faecal LPS levels and suppressing immune responses.

Evaluation of an outbred mouse model for Francisella tularensis vaccine development and testing.

Francisella tularensis (Ft) is a biothreat agent for which there is no FDA-approved human vaccine. Currently, there are substantial efforts underway to develop both vaccines and the tools to assess these vaccines. Tularemia laboratory research has historically relied primarily upon a small number of inbred mouse strains, but the utility of such findings to outbred animals may be limited. Specifically, C57BL/6 mice are more susceptible than BALB/c mice to Ft infection and less easily protected against challenge with highly virulent type A Ft. Thus, depending on the inbred mouse strain used, one could be misled as to which immunogen(s)/vaccine will ultimately be effective in an outbred human population. Accordingly, we evaluated an outbred Swiss Webster (SW) mouse model in direct comparison to a well-established, inbred C57BL/6 mouse model. Mucosal vaccination with the live, attenuated Ft LVS superoxide dismutase (sodB) mutant demonstrated significantly higher protection in outbred SW mice compared to inbred C57BL/6 mice against Ft SchuS4 respiratory challenge. The protection observed in vaccinated outbred mice correlated with lower bacterial density, reduced tissue inflammation, and reduced levels of pro-inflammatory cytokine production. This protection was CD4+ and CD8+ T cell-dependent and characterized by lower titers of serum antibody (Ab) that qualitatively differed from vaccinated inbred mice. Enhanced protection of vaccinated outbred mice correlated with early and robust production of IFN-γ and IL-17A. Neutralizing Ab administered at the time of challenge revealed that IFN-γ was central to this protection, while IL-17A neutralization did not alter bacterial burden or survival. The present study demonstrates the utility of the outbred mouse as an alternative vaccination model for testing tularemia vaccines. Given the limited MHC repertoire in inbred mice, this outbred model is more analogous to the human in terms of immunological diversity.

232 - SOCS1 S-Nitrosation in the nucleus is a switch of macrophage polarization and function

Macrophages play a critical role in inflammation initiation, progression and resolution due to their capacity to assume both pro- and anti-inflammatory phenotypes. The molecular mechanisms that regulate these transitions are of great clinical interest but remain poorly understood. We discovered that suppressor of cytokine signaling-1, an enzyme that targets and degrades RelA, is susceptible to S-nitrosation on cisteines 147 and 179 specifically in the nucleus. Nitrosation in the nucleus is effected by nitric oxide synthase-1 (NOS1) in macrophages. Nitrosation of SOCS1 prevents its interaction with RelA, and the formation of the pro-inflammatory p65-p50 NFkB heterodimer. In NOS1 knockout macrophages, SOCS1 is not nitrosated upon activation of the inflammatory response leading to rapid degradation of RelA/p65 with the accumulation of p50. Accumulation of p50 promotes homodimerization (p50-p50) leading to anti-inflammatory gene transcription. Interestingly, transplantation of NOS1 knockout bone marrow-derived macrophages (BMDM) into the inflammation prone lungs of SOCS1fl/fl.LysMCre mice is protective against LPS-induced tissue injury. In addition, levels of pro-inflammatory cytokine production in these lungs are markedly reduced after LPS challenge suggesting NOS1KO BMDM have the capacity to suppress pro-inflammatory immune polarization and associated tissue injury in a model of chronic inflammatory lung disease. Overall our results indicate that SOCS1 acts as a switch between pro- and anti-inflammatory macrophage polarization regulated by NOS1-mediated nitrosation.

Immunostimulatory effect of Zinc Phthalocyanine derivatives on macrophages based on the pro-inflammatory TNFα and IL1β cytokine production levels

In this study, we examined the effect of halogen substitution on the ability of ZnPcs to stimulate or regulate the immune system cells. There have been studies focusing on the usage of Pcs as treatment option against different cancer types. More attention should be paid on their possible positive or negative effects on the immune system for better prognosis rates. We designed and synthesized unique ZnPcs with iodine substitution and further tested their effect on mammalian macrophage cell line. Macrophages are crucial cell types that can define the immune response by cytokine production as well as by antigen presentation to the other immune system cells after phagocytosis of the danger associated molecules. Our results suggest an immunostimulatory role for the iodine substituted ZnPc on macrophages based on the changes in pro-inflammatory cytokine production levels (TNFα, IL1β and IL6). This effect dependent on the presence of iodine; since non-substituted ZnPc did not exert such a stimulatory activity on the macrophages. These results support a possible use of the reagents against the tumor types that would get affected detrimentally by the pro-inflammatory environment.

Macrophage C-type lectin is essential for phagosome maturation and acidification during Escherichia coli-induced peritonitis

Sepsis is a life-threatening condition caused by an uncontrolled response to bacterial infection. Impaired bactericidal activity in the host is directly associated with severe sepsis; however, the underlying regulatory mechanism(s) is largely unknown. Here, we show that MCL (macrophage C-type lectin) plays a crucial role in killing bacteria during Escherichia coli-induced peritonitis. MCL-deficient mice with E. coli-induced sepsis showed lower survival rates and reduced bacterial clearance when compared with control mice, despite similar levels of proinflammatory cytokine production. Although the ability of macrophages from MCL-deficient mice to kill bacteria was impaired, they showed normal phagocytic activity and production of reactive oxygen species. In addition, MCL-deficient macrophages showed defective phagosome maturation and phagosomal acidification after E. coli infection. Taken together, these results indicate that MCL plays an important role in host defense against E. coli infection by promoting phagosome maturation and acidification, thereby providing new insight into the role of MCL during pathogenesis of sepsis and offering new therapeutic options.

High-mobility group box-1 protein induces osteogenic phenotype changes in aortic valve interstitial cells

ObjectivesCalcific aortic valve (AV) disease is known to be an inflammation-related process. High-mobility group box-1 (HMGB1) protein and Toll-like receptor 4 (TLR4) have been reported to participate in several inflammatory diseases. The purpose of the present study was to determine whether the HMGB1-TLR4 axis is involved in calcific AV disease, and to evaluate the effect of HMGB1, and its potential mechanisms, on the pro-osteogenic phenotype change of valvular interstitial cells (VICs). MethodsExpression of HMGB1 and TLR4 in human calcific AVs was evaluated using immunohistochemical staining and immunoblotting. Cultured VICs were used as an in vitro model. The VICs were stimulated with HMGB1 for analysis, with versus without TLR4 small interfering ribonucleic acid (siRNA), c-Jun N-terminal kinase mitogen-activated protein kinase (JNK MAPK), and nuclear factor kappa-B (NF-κB) inhibitors. ResultsEnhanced accumulation of HMGB1 and TLR4 was observed in calcific valves. Moreover, we found that HMGB1 induced high levels of pro-inflammatory cytokine production and promoted the osteoblastic differentiation and calcification of VICs. In addition, HMGB1 induced phosphorylation of JNK MAPK and NF-κB. However, these effects were markedly suppressed by siRNA silencing of TLR4. In addition, blockade of JNK MAPK and NF-κB phosphorylation prohibited HMGB1-induced production of pro-osteogenic factors, and mineralization of VICs. ConclusionsThe HMGB1 protein may promote osteoblastic differentiation and calcification of VICs, through the TLR4-JNK-NF-κB signaling pathway.

Oxidized Low-Density Lipoprotein Promotes Osteoblastic Differentiation of Valvular Interstitial Cells through RAGE/MAPK

Objectives: We have previously shown that oxidized low-density lipoprotein (oxLDL) promotes the osteogenic differentiation of valvular interstitial cells (VICs) by inducing endoplasmic reticulum (ER) stress. We also demonstrated the detrimental role of the receptor for advanced glycation end products (RAGE) activation and signaling in the development and progression of aortic valve (AV) calcification. Here, we test the hypothesis that oxLDL may induce the osteoblastic differentiation of VICs via RAGE. Methods: Cultured porcine aortic VICs were used in an in vitro model. The VICs were incubated with oxLDL for analysis, with and without RAGE siRNA. Results: We found that oxLDL markedly increased the expression of RAGE, induced high levels of proinflammatory cytokine production and promoted the osteoblastic differentiation and calcification of VICs. oxLDL also induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) MAPK. However, these effects were found to be markedly suppressed by siRNA silencing of RAGE. Conclusions: Our data provide evidence that RAGE mediates oxLDL-induced activation of p38 and JNK MAPK and the osteogenic differentiation of VICs.

Differential expression and functional role of cannabinoid genes in alcohol users

BackgroundGenetic factors account for about fifty percent of the risk for alcoholism and alcohol dependence (AD) has been reported to be influenced by cannabinoid receptors (CBRs) and the endocannabinoid system (ECS). Previous studies have focused on cannabinoids and alcohol-related effects in the CNS; however, the role CBRs play on alcohol effects in the immune system has not been elucidated yet. Since alcohol can affect immune responses and have detrimental effects on immune cells such as dendritic cells (DCs), we hypothesize that alcohol can exert its effects on DCs by modulating changes in CBRs, which in turn can regulate important DC functions such as cytokine production. MethodsTherefore, we studied the expression of CNR1 and CNR2, and the novel cannabinoid G protein-coupled receptor (GPCR) 55 (GPR55) in human monocyte-derived dendritic cells (MDDCs) from alcohol users. CNR1, CNR2, and GPR55 genes were measured by qRT-PCR and protein by flow cytometry. MDDCs from alcohol users show significantly higher levels of CNR2 and GPR55 compared to MDDCs from non-users. These findings were further confirmed using MDDCs treated with alcohol. Inflammatory cytokines were measured in EtOH-treated and non-treated cells by antibody array. ResultsFunctional effects of CBRs on MDDCs were shown by CB2 and GPR55 siRNA transfection. Transfected EtOH-treated cells showed significantly higher levels of proinflammatory cytokine production as measured by IL-1β expression. Our results provide insights into alcohol mechanisms of DC regulation and show, for the first time, that alcohol is inducing CNR2 and GPR55 in human DCs.

Bacterial Cell Wall Constituents Induce Hepcidin Expression in Macrophages Through MyD88 Signaling

Hepcidin is a key regulator of iron recycling by macrophages that is synthesized mainly by hepatocytes but also by macrophages. However, very little is known about the molecular regulation of hepcidin in macrophages. In the present study, we investigated hepcidin regulation in the RAW264.7 macrophage cell line and in murine peritoneal macrophages stimulated with different Toll-like receptor (TLR) ligands. We found that TLR-2 and TLR-4 ligands activated hepcidin expression in RAW264.7 cells and in wild-type murine peritoneal macrophages, but not in murine peritoneal macrophages isolated from TLR2(-/-), TLR-4-deficient or MyD88(-/-) mice. IL-6 production by RAW264.7 cells stimulated with lipopolysaccharide (LPS, TLR4 ligand) was enhanced by high amounts of iron present in the culture medium. We conclude that hepcidin expression in macrophages is regulated mainly through TLR2 and TLR4 receptors via the MyD88-dependent signaling pathway and that autocrine regulation of iron accumulation in macrophages by hepcidin may affect the levels of proinflammatory cytokine production.

Sphingosine Kinase And Sphingosine-1-Phosphate Receptors: Novel Therapeutic Targets Of Rheumatoid Arthritis?

Rheumatoid arthritis (RA) is a chronic, destructive, autoimmune joint disease characterized by elevated levels of proinflammatory cytokine production. Sphingosine kinase (SphK) phosphorylates sphingosine into sphingosine-1-phosphate. Synovial fluid of RA patients exhibits significantly higher levels of S1P than their non-inflammatory osteoarthritis counterparts. SphK blockade suppresses cytokines and MMP-9 release in RA peripheral blood mononuclear cells. In addition, downregulation of SphK1 either through a specific siRNA approach or transgenic human TNF-α SphK1-deficient mice (hTNF-α/SphK1(-/-)) exhibit significantly less synovial inflammation and joint pathology. By contrast, SphK2 modulation leads to disease exacerbation. These results clearly demonstrate that such anti- and proinflammatory potential of SphK1/2 modulation may alter the outcome in RA synovitis and raises the possibility that drugs that specifically target SphK1 activity may play a beneficial role in the treatment of RA and other autoimmune rheumatic diseases.

The elucidation of gene transferring mechanism by ultrasound-responsive unmodified and mannose-modified lipoplexes

The development of gene transfection methods enhancing the level of gene expression under simple and low-toxic condition is required for gene therapy in clinical. Our group has developed the ultrasound (US)-mediated gene transfection method using Man-PEG 2000 bubble lipoplexes, which are US-responsive and mannose-modified gene carriers, and succeeded in obtaining the enhanced gene expression in mannose receptor-expressing cells selectively by the gene transfer using Man-PEG 2000 bubble lipoplexes with US exposure in vitro and in vivo. Here, we investigated pDNA transferring mechanism followed by US exposure to unmodified and Man-PEG 2000 bubble lipoplexes, in particular, focused on US exposure timing. Following investigation of intracellular transferring characteristics, a large amount of pDNA was transferred into the cytoplasm followed by US-mediated destruction of bubble lipoplexes in the gene transfer using both bubble lipoplexes with US exposure. Moreover, the effective gene expression was obtained without TNF-α production when US was exposed until 5 min after the addition of bubble lipoplexes. These findings suggest that the gene transfer using unmodified and Man-PEG 2000 bubble lipoplexes with US exposure enables to transfer pDNA into the cytoplasm, and optimized US exposure timing is important to achieve the high level of gene expression and the low level of pro-inflammatory cytokine production.