Increase In IL-10 Production Research Articles

Intracellular Interaction of Interleukin (IL)-32α with Protein Kinase Cϵ (PKCϵ) and STAT3 Protein Augments IL-6 Production in THP-1 Promonocytic Cells

IL-32α is known as a proinflammatory cytokine. However, several evidences implying its action in cells have been recently reported. In this study, we present for the first time that IL-32α plays an intracellular mediatory role in IL-6 production using constitutive expression systems for IL-32α in THP-1 cells. We show that phorbol 12-myristate 13-acetate (PMA)-induced increase in IL-6 production by IL-32α-expressing cells was higher than that by empty vector-expressing cells and that this increase occurred in a time- and dose-dependent manner. Treatment with MAPK inhibitors did not diminish this effect of IL-32α, and NF-κB signaling activity was similar in the two cell lines. Because the augmenting effect of IL-32α was dependent on the PKC activator PMA, we tested various PKC inhibitors. The pan-PKC inhibitor Gö6850 and the PKCε inhibitor Ro-31-8220 abrogated the augmenting effect of IL-32α on IL-6 production, whereas the classical PKC inhibitor Gö6976 and the PKCδ inhibitor rottlerin did not. In addition, IL-32α was co-immunoprecipitated with PMA-activated PKCε, and this interaction was totally inhibited by the PKCε inhibitor Ro-31-8220. PMA-induced enhancement of STAT3 phosphorylation was observed only in IL-32α-expressing cells, and this enhancement was inhibited by Ro-31-8220, but not by Gö6976. We demonstrate that IL-32α mediated STAT3 phosphorylation by forming a trimeric complex with PKCε and enhanced STAT3 localization onto the IL-6 promoter and thereby increased IL-6 expression. Thus, our data indicate that the intracellular interaction of IL-32α with PKCε and STAT3 promotes STAT3 binding to the IL-6 promoter by enforcing STAT3 phosphorylation, which results in increased production of IL-6.

HIV-1 Is Not a Major Driver of Increased Plasma IL-6 Levels in Chronic HIV-1 Disease

Increased plasma IL-6 levels have been associated with HIV-1 disease progression risk, yet the drivers of IL-6 production in HIV-1 infection are not known. This study was designed to explore the relationship between HIV-1 replication and IL-6 induction. Correlations between plasma levels of IL-6 and HIV-1 RNA were examined in 2 clinical studies. To more directly assess the induction of IL-6 by HIV-1, several cell and tissue types that support HIV-1 replication in vivo were infected with HIV-1, and expression of IL-6 was measured. Spearman rank correlations were used to examine the relationship between plasma levels of IL-6 and HIV-1 RNA. Macrophages and colonic and lymph node histocultures were infected with HIV-1 or stimulated with bacterial products, lipopolysaccharide (LPS) or flagellin, and IL-6 levels in supernatant were measured by enzyme-linked immunosorbent assay or multiplex bead assay. In the clinical studies, there was weak or no correlation between plasma levels of IL-6 and HIV-1 RNA, but IL-6 levels were correlated with plasma levels of the LPS coreceptor CD14. Macrophages stimulated with LPS or flagellin showed robust production of IL-6, but there was no increase in IL-6 production after HIV-1 infection. IL-6 expression was not increased in lymph node histocultures obtained from HIV-1-infected subjects nor after productive HIV-1 infection of colonic or lymph node histocultures ex vivo. We find no evidence that HIV-1 replication is an important driver of IL-6 expression in vivo or in in vitro systems.

Age-related susceptibility and resistance to nonlethal Plasmodium yoelii infection in C57BL/6 mice

In cases of human malaria, children suffer very high rates of morbidity and mortality. To analyse the mechanisms involved in age-dependent protection against malaria, we investigated the characterization of immune responses to Plasmodium yoelii 17XNL (P.y 17XNL) in young (3 weeks) and middle-aged (8 months) C57BL/6 mice. In this study, we found that 100% of young mice succumbed to P.y 17XNL infection with higher parasitemia, while middle-aged mice were able to clear blood parasites and no mortality was observed. These observations suggested that the young C57BL/6 mice were susceptible to P.y 17XNL infection, whereas the middle-aged mice were resistant. Cellular analysis revealed that both the numbers of splenic myeloid dendritic cells (mDCs) as well as the expression of DC maturation markers were higher in middle-aged mice than those in young mice. The numbers of IgG1- or IgG2a-secreting B cells increased markedly in middle-aged mice after infection with P.y 17XNL. The dynamic change of the number of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in mice infected with P.y 17XNL was also different between the two groups. In addition, the levels of IFN-gamma and NO increased in both groups during early parasite infection, while there was also an obvious increase in IL-4 production in the infected middle-aged mice. The change in IL-10 levels following infection was consistent with that of the change in the number of Tregs. The survival of middle-aged mice following P.y 17XNL infection was dependent upon the establishment of effective Thl and Th2 responses and a successful switch between Th1 and Th2 responses, as well as appropriate functioning of Tregs.

The Wnt inhibitor secreted Frizzled‐Related Protein 1 (sFRP1) promotes human Th17 differentiation

Wnt/β-catenin signaling plays a crucial role during embryogenesis and tumorigenesis, and in T cells, promotes the differentiation of Th2 cells. However, the role of Wnt signals in the differentiation and maintenance of human Th17 cells remains poorly understood. We found that the higher levels of IL-17 in the synovial fluid of rheumatoid arthritis (RA) patients compared with that of osteoarthritis (OA) patients were associated with a higher concentration of sFRP1 (secreted Frizzled-Related Protein 1), an inhibitor of the Wnt/β-catenin pathway. The addition of sFRP1 during TCR-mediated stimulation induced a significant increase in IL-17 production by both naïve and memory CD4(+) T cells. Moreover, under Th17-differentiation conditions, the addition of sFRP1 significantly reduced the requirement for TGF-β. Mechanistically, we observed that sFRP1 significantly enhanced the phosphorylation of Smad2/3 in CD4(+) T cells upon TGF-β stimulation and that blocking TGF-β signaling abolished the Th17-promoting activity of sFRP1. Our findings reveal a novel function for sFRP1 as a potent inducer of human Th17-cell differentiation. Consequently, sFRP1 may represent a promising target for the treatment of Th17-mediated disease in humans.

Milk Matters: Soluble Toll-Like Receptor 2 (sTLR2) in Breast Milk Significantly Inhibits HIV-1 Infection and Inflammation

The majority of infants who breastfeed from their HIV-positive mothers remain uninfected despite constant and repeated exposure to virus over weeks to years. This phenomenon is not fully understood but has been closely linked to innate factors in breast milk (BM). Most recently we have focused on one such innate factor, soluble Toll-like receptor 2 (sTLR2) for its significant contribution as an inhibitor of inflammation triggered by bacterial and viral antigens. We hypothesized that sTLR2 in BM inhibits immune activation/inflammation and HIV-1 infection. sTLR2 protein profiles were analyzed in HIV-uninfected BM and showed dramatic variability in expression concentration and predominant sTLR2 forms between women. sTLR2 immunodepleted BM, versus mock-depleted BM, incubated with Pam3CSK4 lead to significant increases in IL-8 production in a TLR2-dependant fashion in U937, HEK293-TLR2, and Caco-2. Importantly, TLR2-specific polyclonal and monoclonal antibody addition to BM prior to cell-free R5 HIV-1 addition led to significantly (P<0.01, P<0.001, respectively) increased HIV-1 infection in TZM-bl reporter cells. To confirm these findings, sTLR2-depletion in BM led to significantly (P<0.001) increased HIV-1 infection in TZM-bl cells. Notably, immunodepletion does not allow for the complete removal of sTLR2 from BM, thus functional testing shown here may underestimate the total effect elicited by sTLR2 against HIV-1 and synthetic bacterial ligand. This study provides evidence for the first time that sTLR2 in BM may provide a dual protective role for infants breastfeeding from their HIV-infected mothers by; (1) immunomodulating pro-inflammatory responses to bacterial ligands, and (2) directly inhibiting cell-free HIV-1 infection. Thus, sTLR2 in BM may be critical to infant health and prove beneficial in decreasing vertical HIV-1 transmission to infants.

Superior molecularly altered influenza virus hemagglutinin peptide 308–317 inhibits collagen‐induced arthritis by inducing CD4+ Treg cell expansion

To investigate the inhibitory effect and possible mechanism of a novel influenza virus hemagglutinin 308-317 peptide (altered HA308-317 peptide) in collagen-induced arthritis (CIA). CIA was induced in DBA/1 mice by immunization with type II collagen (CII). Altered HA308-317 peptide, wild HA308-317 peptide, wild CII263-272 peptide, and irrelevant peptide were administered intranasally beginning at arthritis onset. Clinical and histologic scores were assessed, and cytokine levels were determined in the serum or in supernatants from splenocytes. Characteristics of T cell subsets in response to different peptides were analyzed both in vivo and in vitro. Intranasal administration of wild CII263-272 peptide, wild HA308-317 peptide, or altered HA308-317 peptide could significantly ameliorate CIA, but altered HA308-317 peptide showed greater therapeutic effects than wild CII263-272 peptide and wild HA308-317 peptide. The effect of altered HA308-317 peptide was associated with a substantial decrease in production of interleukin-17 (IL-17) and interferon-γ (IFNγ) and with a marked increase in production of IL-10 and transforming growth factor β, both in serum and in supernatants from splenocytes treated with altered HA308-317 peptide. Both the number and function of CD4+ Treg cells were significantly up-regulated by altered HA308-317 peptide, with a decreased induction of Th1 cells (CD4+IFNγ+) and Th17 cells (CD4+IL-17+). Adoptive transfer of CD4+CD25+ T cells from altered HA308-317 peptide-treated mice resulted in greater suppressive capacity in ameliorating CIA severity than did adoptive transfer of CD4+CD25+ T cells from wild HA308-317 peptide-treated, wild CII263-272 peptide-treated, or irrelevant peptide-treated mice. Intranasal administration of altered HA308-317 peptide potently suppressed the severity of CIA by increasing the number and function of CD4+ Treg cells, suggesting that altered HA308-317 peptide might be a promising candidate for treatment of rheumatoid arthritis.

Divergent Responses of Different Endothelial Cell Types to Infection with Candida albicans and Staphylococcus aureus

Endothelial cells are important in the pathogenesis of bloodstream infections caused by Candida albicans and Staphylococcus aureus. Numerous investigations have used human umbilical vein endothelial cells (HUVECs) to study microbial-endothelial cell interactions in vitro. However, the use of HUVECs requires a constant supply of umbilical cords, and there are significant donor-to-donor variations in these endothelial cells. The use of an immortalized endothelial cell line would obviate such difficulties. One candidate in this regard is HMEC-1, an immortalized human dermal microvascular endothelial cell line. To determine if HMEC-1 cells are suitable for studying the interactions of C. albicans and S. aureus with endothelial cells in vitro, we compared the interactions of these organisms with HMEC-1 cells and HUVECs. We found that wild-type C. albicans had significantly reduced adherence to and invasion of HMEC-1 cells as compared to HUVECs. Although wild-type S. aureus adhered to and invaded HMEC-1 cells similarly to HUVECs, an agr mutant strain had significantly reduced invasion of HMEC-1 cells, but not HUVECs. Furthermore, HMEC-1 cells were less susceptible to damage induced by C. albicans, but more susceptible to damage caused by S. aureus. In addition, HMEC-1 cells secreted very little IL-8 in response to infection with either organism, whereas infection of HUVECs induced substantial IL-8 secretion. This weak IL-8 response was likely due to the anatomic site from which HMEC-1 cells were obtained because infection of primary human dermal microvascular endothelial cells with C. albicans and S. aureus also induced little increase in IL-8 production above basal levels. Thus, C. albicans and S. aureus interact with HMEC-1 cells in a substantially different manner than with HUVECs, and data obtained with one type of endothelial cell cannot necessarily be extrapolated to other types.

Effects of aging on endotoxin tolerance induced by lipopolysaccharides derived from Porphyromonas gingivalis and Escherichia coli.

BackgroundPeriodontitis is a bacterially induced chronic inflammatory disease. Exposure of the host to periodontal pathogens and their virulence factors induces a state of hyporesponsiveness to subsequent stimulations, termed endotoxin tolerance. Aging has a profound effect on immune response to bacteria challenge. The aim of this study was to explore the effects of aging on endotoxin tolerance induced by Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) and Escherichia coli (E. coli) LPS in murine peritoneal macrophages.Methodology/Principal FindingsWe studied the cytokine production (TNF-αand IL-10) and Toll-like receptor 2, 4 (TLR2, 4) gene and protein expressions in peritoneal macrophages from young (2-month-old) and middle-aged (12-month-old) ICR mice following single or repeated P. gingivalis LPS or E. coli LPS stimulation. Pretreatment of peritoneal macrophages with P. gingivalis LPS or E. coli LPS resulted in a reduction in TNF-α production and an increase in IL-10 production upon secondary stimulation (p<0.05), and the markedly lower levels of TNF-α and higher levels of IL-10 were observed in macrophages from young mice compared with those from middle-aged mice (p<0.05). In addition, LPS restimulations also led to the significantly lower expression levels of TLR2, 4 mRNA and protein in macrophages from young mice (p<0.05).Conclusions/SignificanceRepeated LPS stimulations triggered endotoxin tolerance in peritoneal macrophages and the ability to develop tolerance in young mice was more excellent. The impaired ability to develop endotoxin tolerance resulted from aging might be related to TLR2, 4 and might lead to the incontrollable periodontal inflammation in older adults.

An In Situ Anti-Inflammatory Drug Delivery System for Intervertebral Disks

IntroductionIntervertebral disk (IVD) degeneration is one of the major causes of low back pain and the exact reason why (and if) it relates with pain symptoms remains to be discovered (Prithvi Raj, 2008). Current conservative treatments include exercise, medications, and physical therapy. Moreover, there is a growing consensus that surgery, towards removing and/or replacing the damaged tissue, may not be effective.This work focuses on the development of a local and minimally invasive anti-inflammatory drug delivery system for IVD. This drug delivery system is based on the incorporation of Chitosan (Ch)/g-Poly(glutamic acid) (PGA) nanocarriers with Diclofenac (Df), a widely used anti-inflammatory drug, in a hyaluronic acid (HA)-based hydrogel. The Df local delivery in IVD would overcome the disadvantages associated with its oral administration such as poor biological half-life, high doses, and side effects such as peptic ulceration. Moreover, the use of a hydrogel envisages the treatment of degenerated IVD, which could be replaced by a novel cell-free or cell-loaded biocompatible gel.Materials and MethodsThe Ch/PGA nanocarriers with Df were produced by coacervation method. Briefly, PGA and Ch electrostatic interaction at a controlled pH (pH 5) was explored as previously described (Antunes, 2012). The nanoparticles obtained were characterized in terms of size and polydispersion index by dynamic light scattering (DLS) and the Df entrapment was evaluated by UV/Vis absorbance. As a vehicle, a commercially available hydrogel based on HA and Gelatin (HyStem, Glycosan) was used. The gelation of the HA-based gel with the nanoparticles was evaluated at different temperatures and dilutions while the gel net was verified using CryoSEM. The quantity and distribution of the nanoparticles within the gel were characterized by fluorimetry and Confocal Microscopy (using FITC-Ch). The Df release from the nanoparticles within the gel was evaluated at physiological pH by UV/Vis absorbance. In the end, the biological effect of this anti-inflammatory strategy was assessed using Lipopolysaccharide (LPS)-induced macrophages isolated from peripheral blood samples. Cell viability, phenotypic markers, and the levels of diverse pro- and anti-inflammatory cytokines were evaluated (IL-6, TNF-alfa, IL-10, etc.).ResultsDf entrapment within Ch/PGA nanoparticles was optimized, reaching approximately 93% of drug entrapment with complexes with size of 253 ± 32 nm. These nano complexes remain stable during several days at pH 5.0 and were successfully incorporated into HA-based hydrogels, being distributed along the gel net as observed by CryoSEM and Confocal Microscopy, despite some aggregates. The gelation of the HA-based gel was optimized: the time of gelation range 25 minutes for gels with 10% of nanoparticles (% v/v), at 37 °C. The decrease of temperature (until 4°C) was shown to increase time of gelation (until 45 minutes).The Df delivery from the nanocomplexes entrapped in the HA-based hydrogel was determined at pH 7.4 (PBS) and compared with Df delivery from free nanoparticles. In this case, an immediate release of the drug occurs (∼ 60% after 1 hour incubation), accomplished by particles aggregation. Nevertheless, the Df release rate was hindered by incorporation of the Df-nanoparticles within the HA hydrogel: after 2 h only 26% of the anti-inflammatory drug was released reaching 100% after 24 h. The Df release in a degenerated disk environment (pH 6.5) is currently being evaluated.The cytotoxic effect of this delivery system is being evaluated in LPS-activated human macrophages. Df, in the range of concentrations used, did not cause any toxicity, while cell metabolic activity did not decrease below 80% in the presence of the nanoparticles. The Df seems to promote the expression of CD 86 and MHC II, in CD14+ macrophages, suggesting a polarization towards M1 or M2b phenotype and an increase in IL-10 production. The levels of prostaglandin (PGE2) are also being determined, since PGE2 is inhibited by anti-inflammatory drugs as Df.ConclusionThe self-assembly of PGA, Ch, and Df resulted in stable nanosystems which can be used for the controlled release of the drug. Sustained Df release at physiological pH is provided by the incorporation of these NPs into an injectable gel. This work shows the feasibility of an in situ anti-inflammatory drug delivery system, controlled by pH changes, for IVD.I confirm having declared any potential conflict of interest for all authors listed on this abstractYesDisclosure of InterestNone declaredPrithvi Raj P. Intervertebral disc: Anatomy-Physiology-Pathophisiology-Treatment. Pain Practice 2008;8(1):18–44Antunes JC, et al. Layer-by-layer self-assembly of Chitosan and Poly(γ-glutamic acid) into polyelectrolyte complexes. Biomacromolecules 2012 In press

An in Situ Anti-Inflammatory Drug Delivery System for Intervertebral Disks

Introduction Intervertebral disk (IVD) degeneration is one of the major causes of low back pain and the exact reason why (and if) it relates with pain symptoms remains to be discovered (Prithvi Raj, 2008). Current conservative treatments include exercise, medications, and physical therapy. Moreover, there is a growing consensus that surgery, towards removing and/or replacing the damaged tissue, may not be effective. This work focuses on the development of a local and minimally invasive anti-inflammatory drug delivery system for IVD. This drug delivery system is based on the incorporation of Chitosan (Ch)/g-Poly(glutamic acid) (PGA) nanocarriers with Diclofenac (Df), a widely used anti-inflammatory drug, in a hyaluronic acid (HA)-based hydrogel. The Df local delivery in IVD would overcome the disadvantages associated with its oral administration such as poor biological half-life, high doses, and side effects such as peptic ulceration. Moreover, the use of a hydrogel envisages the treatment of degenerated IVD, which could be replaced by a novel cell-free or cell-loaded biocompatible gel. Materials and Methods The Ch/PGA nanocarriers with Df were produced by coacervation method. Briefly, PGA and Ch electrostatic interaction at a controlled pH (pH 5) was explored as previously described (Antunes, 2012). The nanoparticles obtained were characterized in terms of size and polydispersion index by dynamic light scattering (DLS) and the Df entrapment was evaluated by UV/Vis absorbance. As a vehicle, a commercially available hydrogel based on HA and Gelatin (HyStem, Glycosan) was used. The gelation of the HA-based gel with the nanoparticles was evaluated at different temperatures and dilutions while the gel net was verified using CryoSEM. The quantity and distribution of the nanoparticles within the gel were characterized by fluorimetry and Confocal Microscopy (using FITC-Ch). The Df release from the nanoparticles within the gel was evaluated at physiological pH by UV/Vis absorbance. In the end, the biological effect of this anti-inflammatory strategy was assessed using Lipopolysaccharide (LPS)-induced macrophages isolated from peripheral blood samples. Cell viability, phenotypic markers, and the levels of diverse pro- and anti-inflammatory cytokines were evaluated (IL-6, TNF-alfa, IL-10, etc.). Results Df entrapment within Ch/PGA nanoparticles was optimized, reaching approximately 93% of drug entrapment with complexes with size of 253 ± 32 nm. These nano complexes remain stable during several days at pH 5.0 and were successfully incorporated into HA-based hydrogels, being distributed along the gel net as observed by CryoSEM and Confocal Microscopy, despite some aggregates. The gelation of the HA-based gel was optimized: the time of gelation range 25 minutes for gels with 10% of nanoparticles (% v/v), at 37 °C. The decrease of temperature (until 4°C) was shown to increase time of gelation (until 45 minutes). The Df delivery from the nanocomplexes entrapped in the HA-based hydrogel was determined at pH 7.4 (PBS) and compared with Df delivery from free nanoparticles. In this case, an immediate release of the drug occurs (∼ 60% after 1 hour incubation), accomplished by particles aggregation. Nevertheless, the Df release rate was hindered by incorporation of the Df-nanoparticles within the HA hydrogel: after 2 h only 26% of the anti-inflammatory drug was released reaching 100% after 24 h. The Df release in a degenerated disk environment (pH 6.5) is currently being evaluated. The cytotoxic effect of this delivery system is being evaluated in LPS-activated human macrophages. Df, in the range of concentrations used, did not cause any toxicity, while cell metabolic activity did not decrease below 80% in the presence of the nanoparticles. The Df seems to promote the expression of CD 86 and MHC II, in CD14+ macrophages, suggesting a polarization towards M1 or M2b phenotype and an increase in IL-10 production. The levels of prostaglandin (PGE2) are also being determined, since PGE2 is inhibited by anti-inflammatory drugs as Df. Conclusion The self-assembly of PGA, Ch, and Df resulted in stable nanosystems which can be used for the controlled release of the drug. Sustained Df release at physiological pH is provided by the incorporation of these NPs into an injectable gel. This work shows the feasibility of an in situ anti-inflammatory drug delivery system, controlled by pH changes, for IVD. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Prithvi Raj P. Intervertebral disc: Anatomy-Physiology-Pathophisiology-Treatment. Pain Practice 2008;8(1):18–44 Antunes JC, et al. Layer-by-layer self-assembly of Chitosan and Poly(γ-glutamic acid) into polyelectrolyte complexes. Biomacromolecules 2012 In press

IFNbeta-1b alters the composition of circulating B cell subsets, and leads to changes in the B cell cytokine secretion profile in patients with relapsing-remitting multiple sclerosis. (116.9)

Abstract Recent studies provide strong evidence for a role for B cells in the pathology of RRMS. In order to explore the effect of IFNbeta-1b treatment on B cell phenotype and function in RRMS patients, blood was drawn from 10 RRMS patients before treatment and again 2 and 6 months after daily injections of IFNbeta-1b. Cryopreserved PBMCs were thawed and stained with panels of antibodies against B cell surface antigens. At baseline, RRMS patients have increased frequencies of naïve CD19+CD20+ B cells and decreased frequencies of memory B cells when compared with age-matched healthy controls. CpG-stimulated PBMCs from patients treated with IFNbeta-1b show an increase in IL-10 production and a decrease in IL-6 production by naïve, memory and B1 cells (a recently-described subset of autoantibody producing cells that are CD20+CD27+CD43+). These changes in cytokine production are indicative of a change from a pro- to an anti-inflammatory phenotype. In addition, this treatment alters the composition of circulating B cell subsets, leading to an increase after six months in circulating naïve B cells and a decrease in both memory and B1 cells, both cell types of which are potentially pathogenic in RRMS. Although the number of subjects in this study was limited, these findings suggest that alterations in B cell phenotype and function may be a mechanism by which IFNbeta-1b reduces disease activity.

Compound A, a Dissociated Glucocorticoid Receptor Modulator, Inhibits T-bet (Th1) and Induces GATA-3 (Th2) Activity in Immune Cells

BackgroundCompound A (CpdA) is a dissociating non-steroidal glucocorticoid receptor (GR) ligand which has anti-inflammatory properties exerted by down-modulating proinflammatory gene expression. By favouring GR monomer formation, CpdA does not enhance glucocorticoid (GC) response element-driven gene expression, resulting in a reduced side effect profile as compared to GCs. Considering the importance of Th1/Th2 balance in the final outcome of immune and inflammatory responses, we analyzed how selective GR modulation differentially regulates the activity of T-bet and GATA-3, master drivers of Th1 and Th2 differentiation, respectively.ResultsUsing Western analysis and reporter gene assays, we show in murine T cells that, similar to GCs, CpdA inhibits T-bet activity via a transrepressive mechanism. Different from GCs, CpdA induces GATA-3 activity by p38 MAPK-induction of GATA-3 phosphorylation and nuclear translocation. CpdA effects are reversed by the GR antagonist RU38486, proving the involvement of GR in these actions. ELISA assays demonstrate that modulation of T-bet and GATA-3 impacts on cytokine production shown by a decrease in IFN-γ and an increase in IL-5 production, respectively.ConclusionsTaken together, through their effect favoring Th2 over Th1 responses, particular dissociated GR ligands, for which CpdA represents a paradigm, hold potential for the application in Th1-mediated immune disorders.

A pilot study of the immunological effects of high-dose vitamin D in healthy volunteers

Although vitamin D deficiency is considered an environmental factor in multiple sclerosis (MS), the immunological and clinical effects of vitamin D supplementation remain unclear. We performed a pilot study of the immunomodulatory effects of vitamin D in healthy individuals (n=4), who took 5000–10,000 IU/day of vitamin D over 15 weeks. After 15 weeks of vitamin D supplementation, serum 25(OH) vitamin D levels rose significantly from baseline, with a corresponding increase in IL-10 production by peripheral blood mononuclear cells and a reduced frequency of Th17 cells. These data provide a strong rationale for randomised trials to assess the clinical effects of vitamin D supplementation in MS.

PTEN in antigen presenting cells is a master regulator for Th17-mediated autoimmune pathology

Autoreactive T cells are a central element in many systemic autoimmune diseases. The generation of these pathogenic T cells is instructed by antigen presenting cells. However, signalling pathways in APC that drive autoimmunity are not completely understood. Here we show that that conditional deletion of PTEN in myeloid cells are almost completely protected from the development of two prototypic model autoimmune diseases, collagen induced arthritis (CIA) and experimental autoimmune encephalomyelitis (EAE). Myeloid specific deletion of PTEN lead to a significant reduction of cytokines pivotal for the induction of systemic autoimmunity such as IL-23 and IL-6 in vitro and in vivo. In addition, PTEN deficient dendritic cells showed reduced activation of p38 MAP-kinase and increased inhibitory phosphorylation of GSK3β in vitro. Dendritic cell and macrophage phenotypic maturation and migration to lymph nodes as well as collagen specific T and B cell activation was comparable in wt and myeloid specific PTEN-/-. However, analysing the impact of myeloid specific PTEN deficiency on T cell polarization, we found a significant reduction of a Th17 type of immune response characterized by reduced production of IL-17 and IL-22. Moreover, there was an increase in IL-4 production and higher numbers of regulatory T cells myeloid specific PTEN-/-. In contrast, myeloid specific PTEN deficiency did not affect serum transfer arthritis, which is independent of the adaptive immune system and solely depends on innate effector functions. These data demonstrate that the presence of PTEN in myeloid cells is required for the development of systemic autoimmunity. Deletion of PTEN in myeloid cells inhibits the development of CIA and EAE by preventing the generation of a pathogenic Th17 type of immune response.

Immunosuppressive DX5+ T cells are potent inhibitors of Th-1 responses via modulation of DCs

Backgroundand objectivesDX5+CD4+ T cells have been shown to have both a protective- and therapeutic effect on collagen-induced arthritis. This protective effect was associated with an increase in IL-10 production. To...

Effects of Yellow Brain Culinary-Medicinal Mushroom, Tremella mesenterica Ritz.:Fr. (Higher Basidiomycetes), on Immune Function in Normal and Type 1 Diabetic Rats

Diabetes mellitus (DM) is characterized by systemic low-grade inflammation and altered immunity. The fruiting bodies (FB) of Tremella mesenterica have been demonstrated to have anti-hyperglycemic and immunomodulatory activities. It is unclear whether submerged culture yeast-like cells (CC) of T. mesenterica have the same immune effects as FB. Here, we compared the immune effects of T. mesenterica FB and CC on immunocyte function. Male Wistar rats were intravenously injected with saline (normal rats) or streptozotocin (50 mg/kg, DM rats) and orally treated with placebo, FB, or CC (1 g/kg/day) for 2 weeks. Peripheral blood leukocytes and splenocytes were collected. In normal rats, FB and CC ingestion significantly decreased T-suppressor leukocyte numbers and interferon (IFN)-γ production in leukocytes (p < 0.05). In addition, CC treatment significantly decreased mitogen-stimulated tumor necrosis factor (TNF)-α and interleukin (IL)-6 production in leukocytes as well as the numbers of total and B splenocytes. In DM rats, FB significantly alleviated the diabetes-induced decreases in plasma TNF-α levels, T-helper splenocyte numbers, and IL-6 production in T leukocytes, and CC significantly attenuated the decreases in plasma TNF-α, IFN-γ, and IL-6 levels, as well as the increase in IL-6 production in T splenocytes induced by diabetes. Moreover, CC significantly decreased the numbers of T-helper leukocytes and B splenocytes as well as the production of TNF-α by splenocytes and IL-4 by leukocytes in DM rats. In conclusion, our results suggest that T. mesenterica FB and CC may decrease peripheral cell-mediated immunity in normal rats. However, in diabetic rats, FB may increase peripheral cell-mediated immunity, and CC may decrease pro-inflammatory and Th1 cytokine production.

RIAM and RapL, Two Structurally Divergent Rap1 Effectors, Have Distinct Signaling and Functional Roles in TCR-Mediated Activation

Abstract 1118Rap1, a small GTPase of the Ras superfamily, is a regulator of cytoskeletal reorganization, adherens junctions positioning and cell adhesion. The best-studied function of Rap1 is inside-out activation of integrins and cell adhesion. Two newly identified Rap1 effectors, RapL and RIAM, have been implicated in Rap1-mediated inside-out activation of integrins and cell adhesion. However, significant differences in the structure of these molecules indicate that they may regulate distinct signaling events. RIAM has a N-terminal coiled-coil region, central RA and pleckstrin homology (PH) domains, and proline-rich N-terminal and C-terminal regions, with multiple FPPPP motifs capable of interacting with the EVH1 domains of the actin regulatory proteins Ena/VASP and multiple XPPPP motifs interacting with Profilin. RIAM interacts with PLC-γ1 and regulates PLC-γ1 spatiotemporal distribution and activation during TCR-mediated stimulation. RapL has an RBD (Ras binding domain-structurally similar to RA domain) and a C-terminal coiled-coil region and interacts with Rap1 via its RBD domain and its N-terminal region and with the aL subunit of LFA-1 via its C-terminal domain. In the present study we investigated the role of RIAM and RapL in regulating TCR-mediated signaling and IL-2 production. We generated RIAM-knockdown (KD) and RapL-KD Jurkat T cells in which endogenous RIAM and RapL, respectively, were depleted by shRNA. Whereas activation of the extracellular signal regulated kinases MEK1/2 and Erk1/2 was impaired by depletion of RIAM, activation of these kinases was unaffected by depletion of RapL. In contrast, activation of p38 and JNK was unaltered in RIAM-KD cells but was abrogated in RapL-KD cells. RIAM knockdown resulted in impaired activation of PLC-γ1, leading to impaired calcium release and activation of calcium and diacylglycerol-dependent GEFs, RasGRP1 and CalDAG-GEFI, thereby inhibiting activation of Ras and Rap1 and abrogating IL-2 production. In contrast, RapL knockdown did not affect PLC-γ1 activation but resulted in dramatic increase in IL-2 production. Because activation of p38 and JNK has been associated with suppression of IL-2, we examined whether inhibition of these pathways in RapL-KD cells might have a causative role in the increased IL-2 production. Forced expression of enzymatically active p38, JNK, or their combination suppressed IL-2 transcription and protein production to the levels of control T cells. To examine whether these two distinct Rap1 effectors display signal hierarchy or independently mediate their opposing effects on MAPKs and IL-2 transcription, we examined whether elimination of RapL in RIAM-KD T cells might restore their ability to produce IL-2. Elimination of RapL did not restore the ability of RIAM-KD cells to produce IL-2. Taken together our results indicate that although RIAM and RapL can mediate LFA-1 activation downstream of Rap1, these Rap1 effectors regulate distinct signaling events and have opposing roles on IL-2 production upon TCR triggering. Moreover, RIAM-mediated signaling is a pre-requisite for RapL to exert its inhibitory effect on IL-2 production. These results indicate that the selective and temporally regulated interaction of Rap1 with these two distinct effectors might have significant implications on the outcome of TCR triggering by inducing initial activation and subsequent suppression of IL-2 production. Disclosures:No relevant conflicts of interest to declare.

Zoledronic acid inhibits macrophage SOCS3 expression and enhances cytokine production.

Suppressor of cytokine signaling-3 (SOCS3) has multiple functions including inhibition of Janus kinase (Jak) activity, regulation of protein degradation, and suppression of cytokine signaling. SOCS3 modulates macrophage response to cytokines such as IL-6 and leptin that are systemically induced in obesity. Obesity is a suspected risk factor for SOCS3-related pathology such as rheumatoid arthritis and Crohn's disease as well as zoledronic acid (ZA)-induced osteonecrosis of the jaw (ONJ). Thus, understanding the ability of bisphosphonates to modulate SOCS3 is necessary to qualify their contribution to these disorders. ONJ occurs in up to 10% of patients using intravenous bisphosphonates and has an unknown pathogenesis that may be linked to decreased bone turnover, altered vascularity, bacterial invasion, and compromised wound healing. Given the increased risk of ONJ with obesity and importance of macrophages in wound healing, we hypothesized that amino-bisphosphonates could contribute to the pathogenesis of ONJ by regulating macrophage responses to cytokines such as leptin and IL-6. We report that ZA is a novel inhibitor of SOCS3 in primary macrophages and human ONJ biopsy specimens. Inhibition of SOCS3 by ZA resulted in significant increases in IL-6 production. SOCS3 transcription is regulated by nuclear accumulation of phosphorylated-Stat3 (P-Stat3). We found that ZA decreased phosphorylation of Stat3 in a mevalonate-pathway dependent manner. However, restoration of P-Stat3 was not sufficient to correct SOCS3 inhibition. We propose that disruption of macrophage SOCS3 expression by amino-bisphosphonates such as ZA may be a novel contributor to inflammatory phenotypes in obesity and the pathogenesis of ONJ.

CCL18 differentiates dendritic cells in tolerogenic cells able to prime regulatory T cells in healthy subjects

AbstractThe aim of this study was to evaluate the nonchemotactic function of CCL18 on human dendritic cells (DCs). In different protocols of DC differentiation, CCL18 was highly produced, suggesting that it may constitute a mandatory mediator of the differentiation process. Differentiation of monocytes from healthy subjects in the presence of granulocyte-macrophage colony-stimulating factor and CCL18 led to the development of DCs with a semimature phenotype, with intermediate levels of costimulatory and MHC class II molecules, increased CCR7 expression, which induced, in coculture with allogenic naive T cells, an increase in IL-10 production. The generated T cells were able to suppress the proliferation of effector CD4+CD25− cells, through a cytokine-dependent mechanism, and exhibited characteristics of type 1 T regulatory cells. The generation of tolerogenic DCs by CCL18 was dependent on the production of indoleamine 2,3-dioxigenase through an interleukin-10-mediated mechanism. Surprisingly, when DCs originated from allergic patients, the tolerogenic effect of CCL18 was lost in relation with a decreased binding of CCL18 to its putative receptor. This study is the first to define a chemokine able to generate tolerogenic DCs. However, this function was absent in allergic donors and may participate to the decreased tolerance observed in allergic diseases.

COMPARISON OF EFFECTS INDUCED BY FORMALDEHYDE EXPOSURE ON ALVEOLAR AND BRONCHIAL EPITHELIAL CELLS IN VITRO

Background and Aims Exposure to formaldehyde (FA) at environmental concentrations is suspected to be associated to asthma and respiratory disorders. Nevertheless, the physiopathology of this relation is still unclear and experimental data are needed. In vitro experimentation is complementary to epidemiological studies. However experimental procedure parameters need to be adapted, such as the choice of target cells of respiratory tract. The aim of this study was to compare the inflammatory response of FA exposure at environmental level on alveolar and bronchial epithelial cells. Methods Human alveolar (A549) and bronchial (BEAS-2B) epithelial cells were exposed at the air-liquid interface in a Vitrocell exposure module to FA (50 μg/m3) (or air for control) during 30 min. After 24h post-incubation, cellular viability was assessed using LDH release. Two chemokines (IL-8, MCP-1) were assayed in the apical supernatant by ELISA. In addition, to modulate inflammatory response, pre-sensitization was performed using LPS (5 μg/mL) (to mimic a bacterian activation), TNFα (1 ng/mL) or macrophages conditioned media (to mimic the macrophages/epithelial cells cooperation) during 16h before exposure. Results Whatever the cell type, cell viability was unaffected by air or FA exposure compared to cells not exposed at the air-liquid interface. Without presensitization or after LPS presensitization, FA had not effect on A549 or BEAS-2B, compared to air-exposed cells. FA exposure, after TNFα presensitization, resulted in an inflammatory response, an increase of IL-8 production by A549 cells; after macrophage conditioned media presensitization, an increase of IL-8 production by A549 cells, and decrease of MCP-1 production by BEAS-2B cells was observed. Conclusions After presensitization FA exposure at environmental levels provokes an inflammatory response in epithelial cells of the respiratory tract. Moreover, this response is differently modulated depending on the level of the cells in the respiratory tract.