Allogeneic apoptotic thymocyte-stimulated dendritic cells expand functional regulatory T cells

P>Dendritic cells (DCs) play an important role in the clearance of apoptotic cells. The removal of apoptotic cells leads to peripheral tolerance, although their role is still not clear. We show that the uptake of apoptotic thymocytes by DCs converts these cells into tolerogenic DCs resistant to maturation by lipopolysaccharide, modulating the production of interleukin-12 and up-regulating the expression of transforming growth factor-beta(1) latency associated peptide. We also observed that DCs pulsed with apoptotic cells in the allogeneic context were more efficient in the expansion of regulatory T cells (Tregs), and that this expansion requires contact between DCs and the T cell. The Tregs sorted from in vitro culture suppressed the proliferation of splenocytes in vitro in a specific and non-specific manner. In the in vivo model, the transfer of CD4+ CD25- cells to Nude mice induced autoimmunity, with cell infiltrate found in the stomach, colon, liver and kidneys. The co-transfer of CD4+ CD25- and CD4+ CD25+ prevented the presence of cell infiltrates in several organs and increased the total cell count in lymph nodes. Our data indicate that apoptotic cells have an important role in peripheral tolerance via induction of tolerogenic DCs and CD4+ CD25+ Foxp3+ cells that present regulatory functions.

SIGNALING PATHWAYS AND MEDIATORS IN LPS-INDUCED LUNG INFLAMMATION IN DIABETIC RATS: ROLE OF INSULIN

Diabetic patients are more susceptible to infections, and their inflammatory response is impaired. This is restored by insulin treatment. In the present study, we investigated the effect of insulin on LPS-induced signaling pathways and mediators in the lung of diabetic rats. Diabetic male Wistar rats (alloxan, 42 mg/kg i.v., 10 days) and control rats received intratracheal instillation of LPS (750 mu g/0.4 mL) or saline. Some diabetic rats were given neutral protamine Hagedorn insulin (4 IU s.c.) 2 h before LPS. After 6 h, bronchoalveolar lavage was performed for the release of mediators, and lung tissue was homogenized for analysis of LPS-induced signaling pathways. Relative to control rats, diabetic rats exhibited a significant reduction in the LPS-induced phosphorylation of extracellular signal-regulated kinase (64%), p38 (70%), protein kinase B (67%), and protein kinase C alpha (57%) and delta (65%) and in the expression of iNOS (32%) and cyclooxygenase 2 (67%) in the lung homogenates. The bronchoalveolar lavage fluid concentrations of NO (47%) and IL-6 (49%) were also reduced in diabetic rats, whereas the cytokine-induced neutrophil chemoattractant 2 (CINC-2) levels were increased 23%, and CINC-1 was not different from control animals. Treatment of diabetic rats with insulin completely or partially restored all these parameters. In conclusion, data presented show that insulin regulates mitogen-activated protein kinase, phosphatidylinositol 3`-kinase, protein kinase C pathways, expression of the inducible enzymes, cyclooxygenase 2 and iNOS, and levels of IL-6 and CINC-2 in LPS-induced lung inflammation in diabetic rats. These results suggest that the protective effect of insulin in sepsis could be due to modulation of cellular signal transduction factors.

Inactivation of formyltransferase (wbkC) gene generates a Brucella abortus rough strain that is attenuated in macrophages and in mice

Rough mutants of Brucella abortus were generated by disruption of wbkC gene which encodes the formyltransferase enzyme involved in LPS biosynthesis. In bone marrow-derived macrophages the B. abortus Delta wbkC mutants were attenuated, could not reach a replicative niche and induced higher levels of IL-12 and TNF-alpha when compared to parental smooth strains. Additionally, mutants exhibited attenuation in vivo in C57BL/6 and interferon regulatory factor-1 knockout mice. Delta wbkC mutant strains induced lower protective immunity in C56BL/6 than smooth vaccine S19 but similar to rough vaccine RB51. Finally, we demonstrated that Brucella wbkC is critical for LPS biosynthesis and full bacterial virulence. (C) 2010 Elsevier Ltd. All rights reserved.

HPV16 Tumor Associated Macrophages Suppress Antitumor T Cell Responses

Purpose: High-risk human papillomavirus (HPV) is the main etiologic factor for cervical cancer. The severity of HPV-associated cervical lesions has been correlated to the number of infiltrating macrophages. The objective of this work is to characterize the role of tumor-associated macrophages (TAM) on the immune cellular response against the tumor. Experimental Design: We used the HPV16 E6- and E7-expressing TC-1 mouse tumor model to study the effect of TAM on T-cell function in vitro, and depleted TAM, using clodronate-containing liposomes, to characterize its role in vivo. Results: TAM, characterized by the positive expression of CD45, F4/80, and CD11b, formed the major population of infiltrating tumor cells. TAM displayed high basal Arginase I activity, producing interleukin-10 (IL-10); they were resistant to iNOSll activity induction, therefore reversion to M1 phenotype, when stimulated in vitro with lipopolysaccharide/IFN gamma, indicating an M2 phentoype. In cultures of isolated TAM, TAM induced regulatory phenotype, characterized by IL-10 and Foxp3 expression, and inhibited proliferation of CD8 lymphocytes. In vivo, depletion of TAM inhibited tumor growth and stimulated the infiltration of tumors by HPV16 E7(49-57)-specific CD8 lymphocytes, whereas depletion of Gr1(+) tumor-associated cells had no effect. Conclusions: M2-like macrophages infiltrate HPV16-associated tumors causing suppression of antitumor T-cell response, thus facilitating tumor growth. Depletion or phenotype alteration of this population should be considered in immunotherapy strategies.

Oncostatin M is a novel glucocorticoid-dependent neuroinflammatory factor that enhances oligodendrocyte precursor cell activity in demyelinated sites

The innate immune reaction to tissue injury is a natural process, which can have detrimental effects in the absence of negative feedbacks by glucocorticoids (GCs). Although acute lipopolysaccharide (LPS) challenge is relatively harmless to the brain parenchyma of adult animals, the endotoxin is highly neurotoxic in animals that are treated with the GC receptor antagonist RU486. This study investigated the role of cytokines of the gp130-related family in these effects, because they are essential components of the inflammatory process that provide survival signals to neurons. Intracerebral LPS injection stimulated expression of several members of this family of cytokines, but oncostatin M (Osm) was the unique ligand to be completely inhibited by the RU486 treatment. OSM receptor (Osmr) is expressed mainly in astrocytes and endothelial cells following LPS administration and GCs are directly responsible for its transcriptional activation in the presence of the endotoxin. In a mouse model of demyelination, exogenous OSM significantly modulated the expression of genes involved in the mobilization of oligodendrocyte precursor cells (OPCs), differentiation of oligodendrocyte, and production of myelin. In conclusion, the activation of OSM signaling is a mechanism activated by TLR4 in the presence of negative feedback by GCs on the innate immune system of the brain. OSM absence is associated with detrimental effects of LPS, whereas exogenous OSM favors repair response to demyelinated regions. (C) 2010 Elsevier Inc. All rights reserved.

Antiparasitic, antineuroinflammatory, and cytotoxic polyketides from the marine sponge Plakortis angulospiculatus collected in Brazil

Investigation of the bioactive crude extract from the sponge Plakortis angulospiculatus from Brazil led to the isolation of plakortenone (1) as a new polyketide, along with five known polyketides (2-6) previously isolated from other Plakortis sponges. The known polyketides were tested in antileishmanial, antitrypanosomal, antineuroinflammatory, and cytotoxicity assays. The results show that plakortide P (3) is a potent antiparasitic compound, against both Leishmania chagasi and Trypanosona cruzi, and exhibited antineuroinflammatory activity. The known polyketides 2-6 were tested for cytotoxicity against four human cancer cell lines, but displayed only moderate cytotoxic activity.

Kinetics of cellular component in inflammatory response induced by different stimuli in the swim bladder of pacu, Piaractus mesopotamicus Holmberg 1887 (Characidae)

The present study assessed the kinetics of cell accumulation at the site of inflammation induced by thioglycolate, Escherichia coli lipopolysaccharide (LPS) and heat-inactivated Aeromonas hydrophila, in the pacu, Piaractus mesopotamicus (Characidae), swim bladder. A quantitative, as well as qualitative, assessment was done of all the cells present in the exudate at 6, 24, and 48 h (n = 8) after inoculation of inflammatory agents. The results show that the thioglycolate was the irritant to induce higher total inflammatory cell accumulation when compared to the control group, 6 h after insult (P < 0.05). Inoculation of heat-inactivated Aeromonas hydrophila induced progressive accumulation of total inflammatory cells, with cell number peaking after 24 h and being significantly higher than observed in the other groups (P < 0.05). Injection of LPS also induced greater cell accumulation when compared to the control group (P < 0.05), although in lower numbers than those induced by the other two irritants. All irritants injected induced significantly greater accumulation of lymphocytes and thrombocytes when compared to the control group (P < 0.05).

Diapocynin versus apocynin as pretranscriptional inhibitors of NADPH oxidase and cytokine production by peripheral blood mononuclear cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

11-Oxoaerothionin isolated from the marine sponge Aplysina fistularis shows anti-inflammatory activity in LPS-stimulated macrophages

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

TNF-alpha, H2O2 and NO response of peritoneal macrophages to Yersinia enterocolitica O : 3 derivatives

In this study, the effect of Yersinia derivatives on nitric oxide (NO), hydrogen peroxide (H2O2) and tumor necrosis factor-alpha (TNF-alpha) production by murine peritoneal macrophages was investigated. Addition of lipopolysaccharide (LPS) to the macrophage culture resulted in NO production that was dose dependent. on the other hand, bacterial cellular extract (CE) and Yersinia outer proteins (Yops) had no effect on NO production. The possible inhibitory effect of Yops on macrophage cultures stimulated with LPS was investigated. Yops partially inhibited NO production (67.4%) when compared with aminoguanidine. The effects of Yersinia derivatives on H2O2 production by macrophages were similar to those on NO production. LPS was the only derivative that stimulated H2O2 release in a dose-dependent manner. All Yersinia derivatives provoked the production of TNF-alpha, but LPS had the strongest effect, as observed for NO production. CE and Yops stimulated TNF-alpha production to a lesser extent than LPS. The results indicate the possibility that in vivo Yops may aid the evasion of the bacteria from the host defense mechanism by impairing the secretion of NO by macrophages. (C) 2003 Elsevier SAS. All rights reserved.

Oxidation of melatonin and its catabolites, N-1-acetyl-N (2)-formyl-5-methoxykynuramine and N-1-acetyl-5-methoxykynuramine, by activated leukocytes

N-1-acetyl-N-2-formyl-5-methoxykynuramine (AFMK) and N-1-acetyl-5-methoxykynuramine (AMK), two melatonin catabolites, have been described as potent antioxidants. We aimed to follow the kinetics of AFMK and AMK formation when melatonin is oxidized by phorbol myristate acetate (PMA) and lipopolysaccharide (LPS)-activated leukocytes. An HPLC-based method was used for AFMK and AMK determination in neutrophil and peripheral blood mononuclear cell cultures supernatants. Samples were separated isocratically on a C18 reverse-phase column using acetonitrile/H2O (25:75) as the mobile phase. AFMK was detected by fluorescence (excitation 340 nm and emission 460 nm) and AMK by UV-VIS absorbance (254 nm). Activation of neutrophils and mononuclear cells with PMA produces larger amounts of AFMK than activation with LPS, probably due to the lower levels of reactive oxygen species formation and myeloperoxidase (MPO) degranulation that occurs when cells are stimulated with LPS. The concentration of AMK found in the supernatant was about 5-10% (from 18-hr cultures) compared with AFMK. This result may reflect its reactivity. Indeed AMK, but not AFMK, is easily oxidized by activated neutrophils in a MPO and hydrogen peroxide-dependent reaction. In conclusion, we defined a simple procedure for the determination of AFMK and AMK in biological samples and demonstrated the capacity of leukocytes to oxidize melatonin and AMK.

Cytotoxicity of chlorhexidine digluconate to murine macrophages and its effect on hydrogen peroxide and nitric oxide induction

Chlorhexidine, even at low concentrations, is toxic for a variety of eukaryotic cells; however, its effects on host immune cells are not well known. We evaluated in vitro chlorhexidine-induced cytotoxicity and its effects on reactive oxygen/nitrogen intermediate induction by murine peritoneal macrophages. Thioglycollate-induced cells were obtained from Swiss mice by peritoneal lavage with 5 ml of 10 mM phosphate-buffered saline, washed twice and resuspended (10(6) cells/ml) in appropriate medium for each test. Cell preparations contained more than 95% macrophages. The cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay and the presence of hydrogen peroxide (H2O2) and nitric oxide (NO) by the horseradish peroxidase-dependent oxidation of phenol red and Griess reaction, respectively. The midpoint cytotoxicity values for 1- and 24-h exposures were 61.12 ± 2.46 and 21.22 ± 2.44 µg/ml, respectively. Chlorhexidine did not induce synthesis or liberation of reactive oxygen/nitrogen intermediates. When macrophages were treated with various sub-toxic doses for 1 h (1, 5, 10, and 20 µg/ml) and 24 h (0.5, 1, and 5 µg/ml) and stimulated with 200 nM phorbol myristate acetate (PMA) solution, the H2O2 production was not altered; however, the NO production induced by 10 µg/ml lipopolysaccharide (LPS) solution varied from 14.47 ± 1.46 to 22.35 ± 1.94 µmol/l and 13.50 ± 1.42 to 20.44 ± 1.40 µmol/l (N = 5). The results showed that chlorhexidine has no immunostimulating activity and sub-toxic concentrations did not affect the response of macrophages to the soluble stimulus PMA but can interfere with the receptor-dependent stimulus LPS.

TLR4 and CD14 receptors expressed in rat pineal gland trigger NFKB pathway

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Glia-Pinealocyte Network: The Paracrine Modulation of Melatonin Synthesis by Tumor Necrosis Factor (TNF)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cytokines released from blood monocytes and expressed in mucocutaneous lesions of patients with paracoccidioidomycosis evaluated before and during trimethoprim-sulfamethoxazole treatment

Background Mucocutaneous lesions in paracoccidioidomycosis are granulomatous and result from tissue responses to Paracoccidioides brasiliensis, the aetiological agent.Objectives and methods In this study we investigate the expression of tumour necrosis factor (TNF)-alpha, interleukin (IL)-10 and transforming growth factor (TGF)-beta 1 by immunohistochemistry in skin and mucosa lesions from patients with the chronic form of paracoccidioidomycosis, evaluated before and at day 20 of trimethoprim-sulfamethoxazole treatment. Cytokine production by peripheral blood monocytes was also studied by enzyme immunoassay.Results Intense immunostaining for TNF-alpha was detected in mononuclear cells that infiltrated granulomas in all skin and mucosa lesions before treatment simultaneously with low IL-10 granular deposits in these cells. At day 20 of treatment, there was reduced TNF-alpha and IL-10 deposition. Immunoreactive TGF-beta 1 was observed diffusely in the dermis and generally in the cytoplasm of macrophages and giant cells, before treatment, and as increased TGF-beta 1 deposits in the fibrosis area at day 20 of treatment. Peripheral blood monocytes from patients with paracoccidioidomycosis, evaluated before treatment, produced high endogenous levels of TNF-alpha, TGF-beta 1 and IL-10 in relation to healthy controls. Lipopolysaccharide-stimulated monocytes from patients secreted lower levels of TNF-alpha in both periods of evaluation while no impairment in capacity of IL-10 and TGF-beta production was observed.Conclusions Trimethoprim-sulfamethoxazole therapy was effective in decreasing fungal load in the lesions, allowing patient immune response to control the infection leading to the healing of the lesions.