Immunostimulatory Role Research Articles

Glomerular disease: limiting autoimmune tissue injury: ROS and the inflammasome.

Schreiber et al. have identified an unexpected immunoregulatory role of reactive oxygen species in antineutrophil cytoplasmic antibody-associated crescentic glomerulonephritis. Here, we discuss these findings in view of other pathways with well-known immunostimulatory roles in kidney disease that have recently been shown to have additional immunosuppressive effects.

Phosphoethanolamine Decoration of Neisseria gonorrhoeae Lipid A Plays a Dual Immunostimulatory and Protective Role during Experimental Genital Tract Infection

The induction of an intense inflammatory response by Neisseria gonorrhoeae and the persistence of this pathogen in the presence of innate effectors is a fascinating aspect of gonorrhea. Phosphoethanolamine (PEA) decoration of lipid A increases gonococcal resistance to complement-mediated bacteriolysis and cationic antimicrobial peptides (CAMPs), and recently we reported that wild-type N. gonorrhoeae strain FA1090 has a survival advantage relative to a PEA transferase A (lptA) mutant in the human urethral-challenge and murine lower genital tract infection models. Here we tested the immunostimulatory role of this lipid A modification. Purified lipooligosaccharide (LOS) containing lipid A devoid of the PEA modification and an lptA mutant of strain FA19 induced significantly lower levels of NF-κB in human embryonic kidney Toll-like receptor 4 (TLR4) cells and murine embryonic fibroblasts than wild-type LOS of the parent strain. Moreover, vaginal proinflammatory cytokines and chemokines were not elevated in female mice infected with the isogenic lptA mutant, in contrast to mice infected with the wild-type and complemented lptA mutant bacteria. We also demonstrated that lptA mutant bacteria were more susceptible to human and murine cathelicidins due to increased binding by these peptides and that the differential induction of NF-κB by wild-type and unmodified lipid A was more pronounced in the presence of CAMPs. This work demonstrates that PEA decoration of lipid A plays both protective and immunostimulatory roles and that host-derived CAMPs may further reduce the capacity of PEA-deficient lipid A to interact with TLR4 during infection.

The effect of garlic powder on human urinary cytokine excretion.

To evaluate the effects of orally administered dehydrated garlic powder on cytokine excretion in the urinary tract. A total of 60 healthy volunteers, randomized into 3 groups, were given a single oral dose of 1 g or 3 g of dehydrated garlic powder or placebo. Urine samples were obtained 6.0 and 24.0 h after garlic intake and assayed for interleukin-8 (IL-8), interleukin- 12 (IL-12), tumor necrosis factor-alpha (TNF-α), diallyl disulfide (DADS) and diallyl sulfide (DAS). Significant increases in IL-12 levels over baseline were noted in urine samples obtained after oral intake of 1 g and 3 g of garlic powder (P < .001). In the 1 g and 3 g garlic powder treatment groups, time-dependent variations in IL-12 levels over the study period were significantly different from the placebo group (P < .001). In both garlic treatment groups, urinary levels of IL-8 and TNF-α were not significantly different from baseline and placebo levels (P > .017). DADS and DAS were not detected in the urine samples at any time after garlic powder intake. Oral intake of doses of garlic traditionally used for daily supplementation increases urinary levels of IL-12, which is a potent stimulator of T helper cell 1 (Th-1) immune responses. This observation encourages further studies investigating the immunostimulatory role of garlic in the urinary tract.

Enhancement of non-specific immune responses in common carp,Cyprinus carpio, by dietary carotenoids obtained from shrimp exoskeleton

The immunostimulatory role of carotenoid extract from shrimp processing discards was evaluated by feeding common carp fingerlings with a diet containing carotenoid extract for 21 weeks at 100 and 200 mg kg�1 astaxanthin levels. Haemoglobin content was significantly (P < 0.05) higher in the blood of fish fed with carotenoid diet (>8.1 g dL�1) compared with that from fish fed with carotenoid deficient diet (6.86 g dL�1) and also leukocyte counts were higher (P < 0.05). No differences (P > 0.05) were observed in total serum protein, globulin level and albumin-globulin ratio, but albumin content was higher (P < 0.05). Respiratory burst activity was significantly (P < 0.05) higher, the serum lysozyme activity almost doubled and the serum bactericidal activity was significantly increased when the fish were fed with diet containing 200 mg kg�1 of astaxanthin but no significant differences were observed in serum trypsin inhibitory activity. There was a significant (P < 0.05) increase in leukocyte myeloperoxidase activity due to dietary carotenoids. Challenging fish with Aeromonas hydrophila after the feeding period resulted in 50% mortality in the control group while in the group fed with diet containing 100 mg kg�1 astaxanthin, the mortality rate was 15%. No mortality and even symptoms of infection was not observed in the group fed with diet containing 200 mg kg�1 of astaxanthin. The study indicated that carotenoid extract from shrimp processing discards can effectively be used as immunostimulants in aquaculture of carps and dietary carotenoids were found to enhance various immune defence mechanisms and also provide protection against the infection of pathogen A. hydrophila.

Immunostimulatory effect of artificial feed supplemented with indigenous plants on Clarias gariepinus against Aeromonas hydrophila

The antibacterial activity of methanol extracts of Ficus benghalensis (prop-root) and Leucaena leucocephala (pod seed) was evaluated by measurement of zone of inhibition against pathogenic bacteria, Escherichia coli and Aeromonas hydrophila. Control artificial feed and artificial feed supplemented with 5% powder of F. benghalensis and L. leucocephala were prepared. Juvenile Clarias gariepinus were divided into four groups, acclimatized to laboratory conditions and fed with respective feeds for 20 days prior to the experiment. Immunomodulatory response of supplementary feed was studied by challenging the fish intraperitoneally at weekly intervals, with A. hydrophila. One set of fish, not challenged with A. hydrophila was used as a negative control, to analyze any detrimental effect of supplementary feed, while positive control, comprised of challenged fish fed with non-supplemented feed. Other two groups of fish were challenged with A. hydrophila and fed with respective supplementary feeds.Blood was collected on weekly intervals for four weeks and serum samples were analyzed to evaluate the damage of fish by A. hydrophila through liver function tests. The increase in the levels of Serum glutamic oxaloacetic transaminase (SGOT) and Serum Glutamic pyruvate transaminase (SGPT) in positive control group indicated the damage of liver & kidney. However the levels did not change significantly in fish fed with supplementary feeds when compared to negative control group. Nitric oxide, SOD, ALP and lipid peroxidase indicated lower stress levels in these fish compared to positive control. Fish fed with supplementary feed showed increased lysozyme activity and phagocytic index indicating an increase in non-specific immune response. The immunoglobulin levels of in serum were analyzed by homologous sandwich ELISA, which showed higher antibody production in fish fed with supplementary feed.The current study suggests conclusively, immunostimulatory role of F. benghalensis (prop-roots) and L. leucocephala (pod seed) in C. gariepinus when supplemented in artificial feed.

Anticancer and immunostimulatory role of encapsulated tumor antigen containing cobalt oxide nanoparticles

The purpose of this study is to evaluate the prospect of using surface modified cobalt oxide(CoO) nanoparticles as carriers of cancerantigens to human macrophages. N-Phosnomethyliminodiacetic acid (PMIDA) was used for surface modification to overcome the toxic effect of CoO nanoparticles. Here, the phosphonate group of the PMIDA acts as a surface-anchoring agent and the remaining -COOH groups bind nonspecifically with tumor associated antigens. This modification allows the conjugation of human oral carcinoma (KB) cell lysate (CL) as an antigen with PMIDA coated CoO nanoparticles (CL-PMIDA-CoO). Particle characterization was performed by dynamic light scattering, atomic force microscopy, and scanning electron microscopy studies. Fourier transform IR spectroscopy was used to investigate conjugation of the protein with nanoparticles. Protein encapsulation was confirmed by protein gel electrophoresis. Active uptake of antigen-conjugated nanoparticles by macrophages was confirmed by fluorescence microscopy. The antitumor activity of the nanocomplex pulsed macrophages was investigated on a human oral carcinoma cell line (KB) in vitro. The modified nanocomplexes upregulate IFN-γ and TNF-α and induce an anticancer immune response by activating macrophages. The use of TNF-α inhibitor confirmed the ability of the CL-PMIDA-CoO nanocomplex to stimulate TNF-α mediated immunostimulation. CL-PMIDA-CoO nanoparticles efficiently increased the CD4(+) population. Thus, our findings provide insight into the use of PMIDA coated CoO nanoparticles as antigen delivery vehicles.

Lymphocyte-mediated macrophage apoptosis during IL-12 stimulation

In contrast to the well known immunostimulatory roles of IL-12, little has been known about its immunosuppressive roles. In the present study, IL-12-activated lymphocyte-mediated macrophage apoptosis was investigated by employing murine lymphocyte/macrophage cocultures. IL-12-activated lymphocytes and their culture supernatants induced an inducible nitric oxide synthase (iNOS)-mediated nitric oxide (NO) synthesis in macrophages. The NO synthesis was markedly inhibited by blocking antibodies to IFN-γ and TNF-α, suggesting the key role of these lymphocyte cytokines in mediating the NO synthesis. The endogenously produced NO inhibited macrophage proliferation, and induced apoptosis in concordance with the accumulation of p53, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and DR5, and the activation of caspase-3, processes that were inhibited by NG-monomethyl-l-arginine, aminoguanidine (NO synthase inhibitors) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (an NO scavenger). These results were further supported by the findings obtained from the experiments employing IFN-γ-knockout and iNOS-knockout mice. Our study demonstrated a novel, non-contact-dependent mechanism of macrophage suppression by IL-12-activated lymphocytes: induction of growth inhibition and apoptosis of macrophages due to endogenous NO synthesis induced by cytokines secreted from IL-12-activated lymphocytes.

Enhancement of Intestinal Immune Function in Mice by β‐D‐Glucan from Aureobasidium Pullulans ADK‐34

β-Glucans, glucose polymers that are the main constituents of the outer cell walls of micro-organisms such as fungi and yeast, are known to play an immunostimulatory role. We prepared β-glucan (β-(1-3),(1-6)-D-glucan) from an edible cultured fungus through fermentation techniques using a strain of Aureobasidium pullulans ADK-34. The purity of this β-glucan preparation (AP-FBG) was demonstrated to be high through various instrumental analyses. We then examined the effects of AP-FBG on intestinal immune systems. We prepared Peyer's patch (PP) cells and measured interleukin (IL)-5, IL-6, and IgA production in culture media with AP-FBG. We found that both cytokines and IgA increased; furthermore, IL-6 secreted by PP dendritic cells (PPDCs) cultured in the presence of AP-FBG significantly increased. We tested IgA production after oral administration of AP-FBG for 2 weeks and found that AP-FBG tended to promote the production of IgA in the small intestine. Interestingly, we observed a significant increase in IgA production in the small intestines of mice treated with cyclophosphamide (CY; an immunosuppressant) after oral administration of AP-FBG diet compared with CY-treated and control diet mice. Production of IL-6 and IgA by PP cells and IL-6 production by PPDCs in AP-FBG-fed and CY-treated mice also increased. These results demonstrate that AP-FBG has the ability to activate PPDC and induce IL-6 production and IgA secretion in PP cells. These abilities were more clearly expressed when AP-FBG was orally administered in a CY-induced immunosuppressed condition. Therefore, AP-FBG may be a useful ingredient for preparing functional foods with immunomodulatory activities.

Immunostimulatory role of Panax ginseng in irradiated bone marrow transplanted rats

Immunostimulatory role of Panax ginseng in irradiated bone marrow transplanted rats

Kupffer cells express a unique combination of phenotypic and functional characteristics compared with splenic and peritoneal macrophages

The immunostimulatory role of Kupffer cells in various inflammatory liver diseases is still not fully understood. In this study, phenotypic and functional aspects of Kupffer cells from healthy C57BL/6 mice were analyzed and compared with those of splenic and peritoneal macrophages to generate a blueprint of the cells under steady-state conditions. In the mouse liver, only one population of Kupffer cells was identified as F4/80(high)CD11b(low) cells. We observed that freshy isolated Kupffer cells are endocytic and show a relatively high basal ROS content. Interestingly, despite expression of TLR mRNA on Kupffer cells, ligation of TLR4, TLR7/8, and TLR9 resulted in a weak induction of IL-10, low or undetectable levels of IL-12p40 and TNF, and up-regulation of CD40 on the surface. Kupffer cells and splenic macrophages show functional similarities, in comparison with peritoneal macrophages, as reflected by comparable levels of TLR4, TLR7/8, and TLR9 mRNA and low or undetectable levels of TNF and IL-12p40 produced upon TLR ligation. The unique, functional characteristics of Kupffer cells, demonstrated in this study, suggest that Kupffer cells under steady-state conditions are specialized as phagocytes to clear and degrade particulates and only play a limited immunoregulatory role via the release of soluble mediators.

Black tea (Camellia sinensis) decoction shows immunomodulatory properties on an experimental animal model and in human peripheral mononuclear cells.

Background:Black tea (Camellia sinensis) has been used as a daily beverage for time immemorial. Immunomodulatory effects of tea are recognized as it stimulates the proliferation of cultured human peripheral blood mononuclear cells. Anti-inflammatory effects of tea have also been depicted in the available literature. Therefore, we designed this study to examine the potential immunemodulatory and anti-inflammatory activities of black tea in a rat model and in human peripheral mononuclear cells.Aims:The purpose of the study was to determine (1) evaluation of anti-inflammatory effects of black tea on rats, (2) evaluation of immunemodulator effects of black tea on rats, and (3) evaluation of immunemodulator effects of black tea on human peripheral mononuclear cells.Materials and Methods:Black tea decoction (10% and 20%) was prepared. Acute anti-inflammatory activity of tea decoction was evaluated using carrageenan and dextran whereas chronic anti-inflammatory (Immunomodulatory) effects were evaluated in a complete Freunds’ adjuvant-induced arthritis model. Immunostimulatory role was evaluated in cultured human (in vitro) peripheral mononuclear cells (T-lymphocytes) by using methyl thioazolyl tetrazolium (MTT) and Trypan blue assay.Study Design:An experimental study was designed.Results:Black tea decoction (10% and 20%) strength has shown significant anti-inflammatory effects (64.8% and 77% reduction, respectively), on carrageenan-induced acute inflammatory models (rat paw edema) which can be comparable with the standard drug indomethacin (89.1%). In a chronic anti-inflammatory model, black tea decoction (10% and 20%) has shown significant suppressive effects on rat paw edema (38.56% and 69.53%) observed on 21st day. Lymphoproliferative action of tea was evaluated on human peripheral mononuclear cells using an MTT assay where the number of living cells were expressed in terms of optical density at 570 nm. An experiment has shown that black tea increases the maximum number of T-lymphocytes at 72 h with a maximum strength of 20%. Maximum number of viable cells (T-lymphocytes) was observed with black tea at 20% strength at 72 h. The results were expressed as mean ± SD, and the significance was evaluated by Student's t-test versus control, with P < 0.05 implying significance.Conclusions:Taken together, our data indicate that black tea has potential anti-inflammatory and immunomodulatory action and this corroborates with the current trend of tea being promoted as a ‘health drink’.

Oral administration of interferon-α2b-transformed Bifidobacterium longum protects BALB/c mice against coxsackievirus B3-induced myocarditis

Multiple reports have claimed that low-dose orally administered interferon (IFN)-α is beneficial in the treatment of many infectious diseases and provides a viable alternative to high-dose intramuscular treatment. However, research is needed on how to express IFN stably in the gut. Bifidobacterium may be a suitable carrier for human gene expression and secretion in the intestinal tract for the treatment of gastrointestinal diseases. We reported previously that Bifidobacterium longum can be used as a novel oral delivery of IFN-α. IFN-transformed B. longum can exert an immunostimulatory role in mice; however the answer to whether this recombinant B. longum can be used to treat virus infection still remains elusive. Here, we investigated the efficacy of IFN-transformed B. longum administered orally on coxsackie virus B3 (CVB3)-induced myocarditis in BALB/c mice. Our data indicated that oral administration of IFN-transformed B. longum for 2 weeks after virus infection reduced significantly the severity of virus-induced myocarditis, markedly down regulated virus titers in the heart, and induced a T helper 1 cell pattern in the spleen and heart compared with controls. Oral administration of the IFN-transformed B. longum, therefore, may play a potential role in the treatment of CVB3-induced myocarditis.

Abstract C222: Diverting Hsp70 trafficking as a tool for stimulating anticancer immunity.

Abstract The stress-inducible heat shock protein 70 (Hsp70) is normally destined for cytosolic localization. In tumors, Hsp70 is frequently localized to the cell surface and released from cells, unusual features that stimulate the immune system against cancer cells. Interestingly, it has been shown that overexpression of Hsp70 enhances the immunogenicity of tumor cells in animal models. Here we investigated potential mechanisms by which overexpression of Hsp70 in B16 melanoma cells may increase surface expression and release. Flow cytometry measurements revealed an increased cell membrane concentration upon elevated level of intracellular Hsp70. Hsp70 was detectable in subcellular fractions enriched in endosomes/ lysosomes. An accumulation of excess Hsp70 was seen in high Hsp70 expressing cells, in particular in the purified lyosomal fractions. In low expressing cells apparent trafficking of Hsp70 was confined to the endosomal system. In contrast, high intracellular Hsp70 concentration was associated with a predominant lysosomal routing. Remarkable changes were observed in Hsp70 trafficking upon heat stress, a common approach used to facilitate Hsp70 release. In control cells a dramatic depletion of surface-exposed Hsp70 was paralleled with increased endosomal recycling of internalized protein. In contrast, excess intracellular Hsp70 prevented the membrane transport from heat-induced depletion, and allowed transport of surface-exposed Hsp70 to lysosomes secreting their cargo in a soluble form. Therefore excess Hsp70 appears to be decisive in terms of both trafficking to the plasma membrane and stress-induced active release. Altogether, our findings demonstrate a vesicular trafficking system bringing excess intracellular Hsp70 to fulfill its immunostimulatory role. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C222.

The Sustained Influence of Short Term Exposure to a Proprietary Extract of North American Ginseng on the Hemopoietic Cells of the Bone Marrow, Spleen and Blood of Adult and Juvenile Mice

This study assessed the influences of CVT-E002, a proprietary extract of North American ginseng, Panax quinquefolius (Afexa Life Sciences, Inc., Edmonton, AB, Canada), in vivo, on murine hemopoietic and immune cells when administered as a dietary additive. The extract was given daily to young, adult mice for a period of 4 weeks, immediately following which one group was euthanized and the hemopoietic and immune cells of their bone marrow, spleen and blood were assayed for CVT-E002-mediated alterations in any of five cell lineages (lymphocytes, nucleated erythroid cells, granulocytes, immature granuloid precursors and monocytes). Another group of these mice was left for a subsequent 8 weeks on control diet, following which the same organs and cell lineages were analysed. In another study, juvenile mice immediately upon weaning (age: 4 weeks), were subjected to the above protocol, and their organs/cell lineages assayed. The results revealed that CVT-E002 had a long-lasting, positive quantitative effect on the lymphocytes and monocytes, regardless of age at commencement of daily, dietary CVT-E002. CVT-E002 may therefore have a prophylactic disease defense, immunostimulatory role, or potentially, even a therapeutic role.

ArtinM, a d-mannose-binding lectin from Artocarpus integrifolia, plays a potent adjuvant and immunostimulatory role in immunization against Neospora caninum

ArtinM and Jacalin (JAC) are lectins from the jackfruit (Artocarpus integrifolia) that have important role in modulation of immune responses to pathogens. Neospora caninum is an Apicomplexa parasite that causes neuromuscular disease in dogs and reproductive disorders in cattle, with economic impact on the livestock industry. Hence, we evaluated the adjuvant effect of ArtinM and JAC in immunization of mice against neosporosis. Six C57BL/6 mouse groups were subcutaneously immunized three times at 2-week intervals with Neospora lysate antigen (NLA) associated with lectins (NLA+ArtinM and NLA+JAC), NLA, ArtinM and JAC alone, and PBS (infection control). Animals were challenged with lethal dose of Nc-1 isolate and evaluated for morbidity, mortality, specific antibody response, cytokine production by spleen cells, brain parasite burden and inflammation. Our results demonstrated that ArtinM was able to increase NLA immunogenicity, inducing the highest levels of specific total IgG and IgG2a/IgG1 ratio, ex vivo Th1 cytokine production, increased survival, the lowest brain parasite burden, along with the highest inflammation scores. In contrast, NLA+JAC immunized group showed intermediate survival, the highest brain parasite burden and the lowest inflammation scores. In conclusion, ArtinM presents stronger immunostimulatory and adjuvant effect than Jacalin in immunization of mice against neosporosis, by inducing a protective Th1-biased pro-inflammatory immune response and higher protection after parasite challenge.

Natural hemozoin stimulates syncytiotrophoblast to secrete chemokines and recruit peripheral blood mononuclear cells

BackgroundPlacental malaria is associated with local accumulation of parasitized erythrocytes, deposition of the parasite hemoglobin metabolite, hemozoin, and accumulation of mononuclear cells in the intervillous space. Fetal syncytiotrophoblast cells in contact with maternal blood are known to respond immunologically to cytoadherent Plasmodium falciparum-infected erythrocytes, but their responsiveness to hemozoin, a potent pro-inflammatory stimulator of monocytes, macrophages and dendritic cells, is not known. MethodsThe biochemical and immunological changes induced in primary syncytiotrophoblast by natural hemozoin was assessed. Changes in syncytiotrophoblast mitogen-activated protein kinase activation was assessed by immunoblotting and secreted cytokine and chemokine proteins were assayed by ELISA. Chemotaxis of peripheral blood mononuclear cells was assessed using a two-chamber assay system and flow cytometry was used to assess the activation of primary monocytes by hemozoin-stimulated syncytiotrophoblast conditioned medium. ResultsHemozoin stimulation induced ERK1/2 phosphorylation. Treated cells secreted CXCL8, CCL3, CCL4, and tumor necrosis factor and released soluble intercellular adhesion molecule-1. Furthermore, the dependence of the hemozoin responses on ERK1/2 stimulation was confirmed by inhibition of chemokine release in syncytiotrophoblast treated with an ERK pathway inhibitor. Hemozoin-stimulated cells elicited the specific migration of PBMCs, and conditioned medium from the cells induced the upregulation of intercellular adhesion molecule-1 on primary monocytes. ConclusionsThese findings confirm an immunostimulatory role for hemozoin and expand the cell types known to be responsive to hemozoin to include fetal syncytiotrophoblast. The results provide further evidence that syncytiotrophoblast cells can influence the local maternal immune response to placental malaria.

Lipid Motif of a Bacterial Antigen Mediates Immune Responses via TLR2 Signaling

The cross-talk between the innate and the adaptive immune system is facilitated by the initial interaction of antigen with dendritic cells. As DCs express a large array of TLRs, evidence has accumulated that engagement of these molecules contributes to the activation of adaptive immunity. We have evaluated the immunostimulatory role of the highly-conserved outer membrane lipoprotein P6 from non-typeable Haemophilus influenzae (NTHI) to determine whether the presence of the lipid motif plays a critical role on its immunogenicity. We undertook a systematic analysis of the role that the lipid motif plays in the activation of DCs and the subsequent stimulation of antigen-specific T and B cells. To facilitate our studies, recombinant P6 protein that lacked the lipid motif was generated. Mice immunized with non-lipidated rP6 were unable to elicit high titers of anti-P6 Ig. Expression of the lipid motif on P6 was also required for proliferation and cytokine secretion by antigen-specific T cells. Upregulation of T cell costimulatory molecules was abrogated in DCs exposed to non-lipidated rP6 and in TLR2−/− DCs exposed to native P6, thereby resulting in diminished adaptive immune responses. Absence of either the lipid motif on the antigen or TLR2 expression resulted in diminished cytokine production from stimulated DCs. Collectively; our data suggest that the lipid motif of the lipoprotein antigen is essential for triggering TLR2 signaling and effective stimulation of APCs. Our studies establish the pivotal role of a bacterial lipid motif on activating both innate and adaptive immune responses to an otherwise poorly immunogenic protein antigen.

Immunostimulatory Activity of Fucoidan from the Brown Alga Fucus evanescens: Role of Sulfates and Acetates

Fucoidans are natural polysaccharides mainly consisting of sulfated α-L-fucopyranose. A wide range of biological activities has been attributed to fucoidans. However, their immunostimulatory role with respect to structure-activity relationships is still under debate. To address this question, hyposulfated (hypoS), deacetylated (deAc), and both hyposulfated and deacetylated (hypoSdeAc) derivatives of native Fucus evanescens fucoidan were used to stimulate bone marrow–derived dendritic cells (BMDCs) and bone marrow–derived macrophages (BMMs). Native fucoidan induced proinflammatory cytokines (IL-1β, IL-6, IL-12, TNF-α) by murine BMDCs and BMMs. Hyposulfation led to a markedly reduced cytokine secretion by BMDCs (35% to 70% reduction compared to native fucoidan) and BMMs (60% to 90% reduction). Cytokine release was also largely decreased by deacetylation (50% to 60% reduction in BMDCs, 50% to 70% reduction in BMMs compared to native fucoidan). In BMDCs and BMMs stimulated with hypoSdeAc, cytokine production was almost completely abolished. In conclusion, our results indicate the importance of sulfate/acetyl groups for the immunostimulatory activity of fucoidans.

Toll-Like Receptor 3 Signaling Enables Human Esophageal Epithelial Cells to Sense Endogenous Danger Signals Released by Necrotic Cells

The mechanisms by which gastroesophageal reflux disease esophagitis develops are controversial. Although many support the notion that caustic injury leads to reflux esophagitis, others have proposed that reflux esophagitis is caused by esophageal epithelial cytokine-mediated inflammation. We previously demonstrated that Toll-like receptor 3 (TLR3) is highly expressed and functional in the nontransformed human esophageal epithelial cell line EPC2-hTERT. In addition to activation by viral double-stranded RNA, TLR3 can be activated by endogenous mRNA released by necrotic cells. In the present study, we investigated the role of esophageal epithelial TLR3 to sense danger signals released by necrotic esophageal epithelial cells in vitro. Following induction of freeze-thaw necrosis, necrotic EPC2-hTERT cell supernatants (NCS) were used to stimulate EPC2-hTERT monolayers, leading to NF-κB-dependent induction of IL-8 mRNA expression. Responses to self-derived NCS were not observed in transformed gastrointestinal epithelial cell lines, including TE-1 and Caco-2 cells, suggesting that the ability to sense endogenous danger signals is unique to nontransformed esophageal epithelial cells. To determine the immunostimulatory role of epithelial RNA, EPC2-hTERT cells were stimulated with self-derived mRNA, which significantly induced IL-8 mRNA expression. Finally, suppression of TLR3 signaling in a DN-TLR3 cell line, hTERT-ΔTIR-TLR3, led to reduced NCS-induced IL-8 induction by both NCS and mRNA stimulation. Our results demonstrate that human esophageal epithelial cells can sense endogenous danger signals, in part through TLR3 signaling. This supports the concept that epithelial injury plays an inciting role in the pathogenesis of reflux-induced esophagitis, providing important insights into the mechanisms by which epithelial injury leads to inflammation.

Toll-like receptor 3 signaling enables human esophageal epithelial cells to sense endogenous danger signals released by necrotic cells

The mechanisms by which gastroesophageal reflux disease esophagitis develops are controversial. Although many support the notion that caustic injury leads to reflux esophagitis, others have proposed that reflux esophagitis is caused by esophageal epithelial cytokine-mediated inflammation. We previously demonstrated that Toll-like receptor 3 (TLR3) is highly expressed and functional in the nontransformed human esophageal epithelial cell line EPC2-hTERT. In addition to activation by viral double-stranded RNA, TLR3 can be activated by endogenous mRNA released by necrotic cells. In the present study, we investigated the role of esophageal epithelial TLR3 to sense danger signals released by necrotic esophageal epithelial cells in vitro. Following induction of freeze-thaw necrosis, necrotic EPC2-hTERT cell supernatants (NCS) were used to stimulate EPC2-hTERT monolayers, leading to NF-κB-dependent induction of IL-8 mRNA expression. Responses to self-derived NCS were not observed in transformed gastrointestinal epithelial cell lines, including TE-1 and Caco-2 cells, suggesting that the ability to sense endogenous danger signals is unique to nontransformed esophageal epithelial cells. To determine the immunostimulatory role of epithelial RNA, EPC2-hTERT cells were stimulated with self-derived mRNA, which significantly induced IL-8 mRNA expression. Finally, suppression of TLR3 signaling in a DN-TLR3 cell line, hTERT-ΔTIR-TLR3, led to reduced NCS-induced IL-8 induction by both NCS and mRNA stimulation. Our results demonstrate that human esophageal epithelial cells can sense endogenous danger signals, in part through TLR3 signaling. This supports the concept that epithelial injury plays an inciting role in the pathogenesis of reflux-induced esophagitis, providing important insights into the mechanisms by which epithelial injury leads to inflammation.