Impaired dendritic cell differentiation and maturation in the absence of C3

Human monocytes can be differentiated into immature dendritic cells (DCs) in the presence of serum and cytokines. One of the main functions of immature DCs is to capture and process antigens. Following maturation, they differentiate into antigen presenting cells. The role of complement in the differentiation process from monocytes towards immature DCs remains elusive. Here we demonstrate that complement 3 (C3) has a regulatory impact on the expression of specific DC surface molecules and DC-derived cytokine production during DC differentiation. We isolated human adherent peripheral blood mononuclear cells, which were cultured in the presence of GM-CSF plus IL-4 in medium supplemented with normal human serum or C3 deficient serum. The lack of C3 during DC differentiation negatively impacted the expression of C-type lectin receptor DC-SIGN, the antigen presenting molecules HLA-DR and CD1a, and the costimulatory molecules CD80 and CD86. Further, the spontaneous production of IL-6 and IL-12 was reduced in the absence of C3. Moreover, the maturation of immature DCs in response to LPS challenge was impaired in the absence of C3 as evidenced by reduced MHC-II, co-stimulatory molecule expression as well as modulated IL-12 and TNF-alpha production. Collectively, our results provide evidence for a novel role of C3 as a critical cofactor in human DC differentiation and maturation. (C) 2007 Elsevier Ltd. All rights reserved.

A Novel Pathway for Inducible Nitric-oxide Synthase Activation through Inflammasomes

Innate immune recognition of flagellin is shared by transmembrane TLR5 and cytosolic Nlrc4 (NOD-like receptor family CARD (caspase activation recruitment domain) domain containing 4)/Naip5 (neuronal apoptosis inhibitory protein 5). TLR5 activates inflammatory genes through MYD88 pathway, whereas Nlrc4 and Naip5 assemble multiprotein complexes called inflammasomes, culminating in caspase-1 activation, IL-1 beta/IL-18 secretion, and pyroptosis. Although both TLR5 and Naip5/Nlrc4 pathways cooperate to clear infections, little is known about the relative anti-pathogen effector mechanisms operating through each of them. Here we show that the cytosolic flagellin (FLA-BSDot) was able to activate iNOS, an enzyme previously associated with TLR5 pathway. Using Nlrc4- or Naip5-deficient macrophages, we found that both receptors are involved in iNOS activation by FLA-BSDot. Moreover, distinct from extracellular flagellin (FLA-BS), iNOS activation by intracellular flagellin is completely abrogated in the absence of caspase-1. Interestingly, IL-1 beta and IL-18 do not seem to be important for FLA-BSDot-mediated iNOS production. Together, our data defined an additional anti-pathogen effector mechanism operated through Naip5 and Nlrc4 inflammasomes and illustrated a novel signaling transduction pathway that activates iNOS.

Melatonin Protects CD4(+) T Cells from Activation-Induced Cell Death by Blocking NFAT-Mediated CD95 Ligand Upregulation

Over the past 20 y, the hormone melatonin was found to be produced in extrapineal sites, including cells of the immune system. Despite the increasing data regarding the biological effects of melatonin on the regulation of the immune system, the effect of this molecule on T cell survival remains largely unknown. Activation-induced cell death plays a critical role in the maintenance of the homeostasis of the immune system by eliminating self-reactive or chronically stimulated T cells. Because activated T cells not only synthesize melatonin but also respond to it, we investigated whether melatonin could modulate activation-induced cell death. We found that melatonin protects human and murine CD4(+) T cells from apoptosis by inhibiting CD95 ligand mRNA and protein upregulation in response to TCR/CD3 stimulation. This inhibition is a result of the interference with calmodulin/calcineurin activation of NFAT that prevents the translocation of NFAT to the nucleus. Accordingly, melatonin has no effect on T cells transfected with a constitutively active form of NFAT capable of migrating to the nucleus and transactivating target genes in the absence of calcineurin activity. Our results revealed a novel biochemical pathway that regulates the expression of CD95 ligand and potentially other downstream targets of NFAT activation. The Journal of Immunology, 2010, 184: 3487-3494.

Apoptosis of macrophages during pulmonary Mycobacterium bovis infection: correlation with intracellular bacillary load and cytokine levels

P>Apoptosis of macrophages infected with pathogenic mycobacteria is an alternative host defence capable of removing the environment supporting bacterial growth. In this work the influence of virulence and bacterial load on apoptosis of alveolar macrophages during the initial phase of infection by Mycobacterium bovis was investigated. BALB/c mice were infected intratracheally with high or low doses of the virulent (ATCC19274) or attenuated (bacillus Calmette-Guerin Moreau) strains of M. bovis. The frequency of macrophage apoptosis, the growth of mycobacteria in macrophages, and the in situ levels of the cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10) and IL-12 and of the anti-apoptotic protein Bcl-2 were measured at day 3 and day 7 post-infection. An increase of macrophage apoptosis was observed after infection with both strains but the virulent strain induced less apoptosis than the attenuated strain. On the 3rd day after infection with the virulent strain macrophage apoptosis was reduced in the high-dose group, while on the 7th day post-infection macrophage apoptosis was reduced in the low-dose group. Inhibition of apoptosis was correlated with increased production of IL-10, reduced production of TNF-alpha and increased production of Bcl-2. In addition, the production of IL-12 was reduced at points where the lowest levels of macrophage apoptosis were observed. Our results indicate that virulent mycobacteria are able to modulate macrophage apoptosis to an extent dependent on the intracellular bacterial burden, which benefits its intracellular growth and dissemination to adjacent cells.

Anti-inflammatory effect of parenteral fish oil lipid emulsion on human activated mononuclear leukocytes

Background & aim: To compare the effect of fish oil-based (FO) lipid emulsions (LE) for parenteral administration with standard LE and a new FO containing LE composed of four different oils on the antigen presentation and inflammatory variables. Methods: Phytohemagglutinin (PHA) activated human mononuclear leukocytes were cultured with different LE - Control: without LE; SO: soybean oil; SO/FO: soybean and FO (4:1); MCT/SO: medium chain triglycerides and SO (1:1); MCT/SO/FO: MCT/SO and FO (4:1) and SMOF: a new LE containing FO. Cytokine production was evaluated by ELISA, the expression of antigen-presenting and co-stimulatory surface molecules were analyzed by flow cytometry and lymphocyte proliferation was assessed by H(3)-Thymidine incorporation, after tetanus toxoid-induced activation. Results: All LE decreased the HLA-DR and increased CD28 and CD152 expression on monocytes/macrophages and lymphocytes surface (p < 0.05). SO/FO and MCT/SO/FO decreased lymphocyte proliferation (p<0.05). All LE decreased IL-2 product ion, but this effect was enhanced with MCT/SO/FO and SMOF (p < 0.05). MCT/SOTO decreased IL-6 and increased IL-10, whereas SO had the opposite effect (p < 0.05). Conclusion: FO LE inhibited lymphocyte proliferation and had an anti-inflammatory effect. These effects seem to be enhanced when FO is mixed with MCT/SO. SMOF had a neutral impact on lymphocyte proliferation and IL-6 and IL-10 production.

Human monocytes but not dendritic cells are killed by blocking of autocrine cyclooxygenase activity

Dendritic cells (DCs), in peripheral tissues, derive mostly from blood precursors that differentiate into DCs under the influence of the local microenvironment. Monocytes constitute the main known DC precursors in blood and their infiltration into tissues is up-regulated during inflammation. During this process, the local production of mediators, like prostaglandins (PGs), influence significantly DC differentiation and function. In the present paper we show that treatment of blood adherent mononuclear cells with 10 mu M indomethacin, a dose achieved in human therapeutic settings, causes monocytes` progressive death but does not affect DCs viability or cell surface phenotype. This resistance of DCs was observed both for cells differentiated in vitro from blood monocytes and for a population with DCs characteristics already present in blood. This phenomenon could affect the local balance of antigen-presenting cells, influence the induction and pattern of immune responses developed under the treatment with non-steroidal anti-inflammatory drugs and, therefore, deserves further investigation. (C) 2009 Elsevier Inc. All rights reserved.

Toll-like receptor 4 agonists adsorbed to aluminium hydroxide adjuvant attenuate ovalbumin-specific allergic airway disease: role of MyD88 adaptor molecule and interleukin-12/interferon-gamma axis

Background Epidemiological and experimental data suggest that bacteria] lipopolysaccharides (LPS) can either protect from or exacerbate allergic asthma. Lipopolysaccharides trigger immune responses through toll-like receptor 4 (TLR4) that in turn activates two major signalling pathways via either MyD88 or TRIF adaptor proteins. The LPS is a pro-Type 1 T helper cells (Th 1) adjuvant while aluminium hydroxide (alum) is a strong Type 2 T helper cells (Th2) adjuvant, but the effect of the mixing of both adjuvants on the development of lung allergy has not been investigated. Objective We determined whether natural (LPS) or synthetic (ER-803022) TLR4 agonists adsorbed onto alum adjuvant affect allergen sensitization and development of airway allergic disease. To dissect LPS-induced molecular pathways, we used TLR4-, MyD88-, TRIF-, or IL-12/IFN-gamma-deficient mice. Methods Mice were sensitized with subcutaneous injections of ovalbumin (OVA) with or without TLR4 agonists co-adsorbed onto alum and challenged with intranasally with OVA. The development of allergic lung disease was evaluated 24 h after last OVA challenge. Results Sensitization with OVA plus LPS co-adsorbed onto alum impaired in dose-dependent manner OVA-induced Th2-mediated allergic responses such as airway eosinophilia, type-2 cytokines secretion, airway hyper-reactivity, mucus hyper production and serum levels of IgE or IgG1 anaphylactic antibodies. Although the levels of IgG2a, Th1 -affiliated isotype increased, investigation into the lung-specific effects revealed that LPS did not induce a Th1 pattern of inflammation. Lipopolysaccharides impaired the development of Th2 immunity, signaling via TLR4 and MyD88 molecules and via the IL-12/IFN-gamma axis, but not through TRIF pathway. Moreover, the synthetic TLR4 agonists that proved to have a less systemic inflammatory response than LPS also protected against allergic asthma development. Conclusion Toll-like receptor 4 agonists co-adsorbed with allergen onto alum down-modulate allergic lung disease and prevent the development of polarized T cell-mediated airway inflammation.

Poly(lactic acid-glycolic acid) nanoparticles markedly improve immunological protection provided by peptide P10 against murine paracoccidioidomycosis

Background and purpose: The present study reports on the preparation and testing of a sustained delivery system for the immunomodulatory peptide P10 aimed at reducing the in vivo degradation of the peptide and the amount required to elicit a protective immune response against paracoccidioidomycosis. Experimental approach: BALB/c mice were infected with the yeast Paracoccidioides brasiliensis to mimic the chronic form of paracoccidioidomycosis. The animals were treated daily with sulfamethoxazole/trimethoprim alone or combined with peptide P10, either emulsified in Freund`s adjuvant or entrapped in poly(lactic acid-glycolic acid) (PLGA) nanoparticles at different concentrations (1 mu g, 5 mu g, 10 mu g, 20 mu g or 40 mu g center dot 50 mu L-1). Therapeutic efficacy was assessed as fungal burden in tissues and the immune response by quantitative determination of cytokines. Key results: Animals given combined chemotherapy and P10 nanotherapy presented a marked reduction of fungal load in the lungs, compared with the non-treated animals. After 30 days of treatment, P10 entrapped within PLGA (1 mu g center dot 50 mu L-1) was more effective than `free` P10 emulsified in Freund`s adjuvant (20 mu g center dot 50 mu L-1), as an adjuvant to chemotherapy. After treatment for 90 days, the higher doses of P10 entrapped within PLGA (5 or 10 mu g center dot 50 mu L-1) were most effective. Treatment with P10 emulsified in Freund`s adjuvant (20 mu g center dot 50 mu L-1) or P10 entrapped within PLGA (1 mu g center dot 50 mu L-1) were accompanied by high levels of interferon-gamma in lung. Conclusions and implications: Combination of sulfamethoxazole/trimethoprim with the P10 peptide entrapped within PLGA demonstrated increased therapeutic efficacy against paracoccidioidomycosis. P10 incorporation into PLGA nanoparticles dramatically reduced the peptide amount necessary to elicit a protective effect.

Association of Human T Lymphotropic Virus 1 Amplification of Periodontitis Severity with Altered Cytokine Expression in Response to a Standard Periodontopathogen Infection

Background. Periodontal diseases (PDs) are infectious diseases in which periodontopathogens trigger chronic inflammatory and immune responses that lead to tissue destruction. Recently, viruses have been implicated in the pathogenesis of PDs. Individuals infected with human T lymphotropic virus 1 (HTLV-1) present with abnormal oral health and a marked increased prevalence of periodontal disease. Methods. In this study, we investigated the patterns of periodontopathogen infection and local inflammatory immune markers in HTLV-1-seropositive individuals with chronic periodontitis (CP/HTLV-1 group) compared with HTLV-1 -seronegative individuals with chronic periodontitis (CP group) and periodontally healthy, HTLV-1 -seronegative individuals (control group). Results. Patients in the CP/HTLV-1 group had significantly higher values of bleeding on probing, mean probing depth, and attachment loss than patients in the CP group. The expression of tumor necrosis factor a and interleukin (IL) 4 was found to be similar in the CP and CP/HTLV-1 groups, whereas IL-12 and IL-17 levels trended toward a higher expression in the CP/HTLV-1 group. A significant increase was seen in the levels of IL-1 beta and interferon gamma in the CP/HTLV-1 group compared with the CP group, whereas expression of the regulatory T cell marker FOXp3 and IL-10 was significantly decreased in the lesions from the CP/HTLV-1 group. Interestingly, similar frequency and/or load of periodontopathogens (Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and Aggregatibacter actinomycetemcomitans) and frequency of viruses (herpes simplex virus 1, human cytomegalovirus, and Epstein-Barr virus) characteristically associated with PDs were found in the CP/HTLV and CP groups. Conclusions. HTLV-1 may play a critical role in the pathogenesis of periodontal disease through the deregulation of the local cytokine network, resulting in an exacerbated response against a standard periodontopathogen infection.

Inhibitory Signals Mediated by Programmed Death-1 Are Involved With T-Cell Function in Chronic Periodontitis

Background: Inhibitory signals mediated via molecules such as programmed death-1 (PD-1) play a critical role in downmodulating immune responses and maintaining peripheral tolerance. We investigated the involvement of cytokines and PD-1 engagement in mediating the T-cell unresponsiveness to bacterial and ubiquitous antigens in periodontal diseases. Methods: Gingival and peripheral blood samples from healthy individuals and patients with chronic periodontitis were collected and used for the subsequent assays. Leukocytes in the lesion site and blood were evaluated using flow cytometry. The production of interferon-gamma, interleukin-10, and transforming growth factor-P proteins was evaluated by enzyme-linked immunosorbent assay (ELISA), and the presence of PD-1+cells in the inflamed gingiva was confirmed by immunofluorescence confocal microscopy for CD4 and PD-1 colocalization. Results: T cells from patients with chronic periodontitis proliferated poorly in response to Aggregatibacter actinomycetem comitans (previously Actinobacillus actinomycetemcomitans) antigen. T-cell unresponsiveness was not associated with imbalanced cytokine production. However, T cells from patients with chronic periodontitis expressed significantly higher levels of PD-1 either upon isolation or after culture with antigens. Moreover, PD-1 blocking did not result in significant T-cell proliferation in cells cultured with phytohemagglutinin or bacterial antigens. The blockade of PD-1 resulted in the increased production of IFN-gamma. In addition, CD4+ and CD8+ T cells expressing PD-1 accumulated in lesions with chronic periodontitis. Conclusion: These data show that PD-1 engagement could be involved in the modulation of IFN-gamma production by T cells in patients with chronic periodontitis. J Periodontol 2009,80:1833-1844.

Distinctive Immunomodulatory and Inflammatory Properties of the Escherichia coli Type II Heat-Labile Enterotoxin LT-IIa and Its B Pentamer following Intradermal Administration

The type I and type II heat-labile enterotoxins (LT-I and LT-II) are strong mucosal adjuvants when they are coadministered with soluble antigens. Nonetheless, data on the parenteral adjuvant activities of LT-II are still limited. Particularly, no previous study has evaluated the adjuvant effects and induced inflammatory reactions of LT-II holotoxins or their B pentameric subunits after delivery via the intradermal (i.d.) route to mice. In the present report, the adjuvant and local skin inflammatory effects of LT-IIa and its B subunit pentamer (LT-IIaB(5)) were determined. When coadministered with ovalbumin (OVA), LT-IIa and, to a lesser extent, LT-IIaB(5) exhibited serum IgG adjuvant effects. In addition, LT-IIa but not LT-IIaB(5) induced T cell-specific anti-OVA responses, particularly in respect to induction of antigen-specific cytotoxic CD8(+) T cell responses. LT-IIa and LT-IIaB(5) induced differential tissue permeability and local inflammatory reactions after i.d. injection. Of particular interest was the reduced or complete lack of local reactions, such as edema and tissue induration, in mice i.d. inoculated with LT-IIa and LT-IIaB(5), respectively, compared with mice immunized with LT-I. In conclusion, the present results show that LT-IIa and, to a lesser extent, LT-IIaB(5) exert adjuvant effects when they are delivered via the i.d. route. In addition, the low inflammatory effects of LT-IIa and LT-IIaB(5) in comparison to those of LT-I support the usefulness of LT-IIa and LT-IIaB(5) as parenterally delivered vaccine adjuvants.

Paracoccidioides brasiliensis Vaccine Formulations Based on the gp43-Derived P10 Sequence and the Salmonella enterica FliC Flagellin

Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. Anti-PCM vaccine formulations based on the secreted fungal cell wall protein (gp43) or the derived P10 sequence containing a CD4(+) T-cell-specific epitope have shown promising results. In the present study, we evaluated new anti-PCM vaccine formulations based on the intranasal administration of P. brasiliensis gp43 or the P10 peptide in combination with the Salmonella enterica FliC flagellin, an innate immunity agonist binding specifically to the Toll-like receptor 5, in a murine model. BALB/c mice immunized with gp43 developed high-specific-serum immunoglobulin G1 responses and enhanced interleukin-4 (IL-4) and IL-10 levels. On the other hand, mice immunized with recombinant purified flagellins genetically fused with P10 at the central hypervariable domain, either flanked or not by two lysine residues, or the synthetic P10 peptide admixed with purified FliC elicited a prevailing Th1-type immune response based on lung cell-secreted type 1 cytokines. Mice immunized with gp43 and FliC and intratracheally challenged with P. brasiliensis yeast cells had increased fungal proliferation and lung tissue damage. In contrast, mice immunized with the chimeric flagellins and particularly those immunized with P10 admixed with FliC reduced P. brasiliensis growth and lung damage. Altogether, these results indicate that S. enterica FliC flagellin modulates the immune response to P. brasiliensis P10 antigen and represents a promising alternative for the generation of anti-PCM vaccines.

Heme Impairs Prostaglandin E(2) and TGF-beta Production by Human Mononuclear Cells via Cu/Zn Superoxide Dismutase: Insight into the Pathogenesis of Severe Malaria

In many hemolytic disorders, such as malaria, the release of free heme has been involved in the triggering of oxidative stress and tissue damage. Patients presenting with severe forms of malaria commonly have impaired regulatory responses. Although intriguing, there is scarce data about the involvement of heme on the regulation of immune responses. In this study, we investigated the relation of free heme and the suppression of anti-inflammatory mediators such as PGE(2) and TGF-beta in human vivax malaria. Patients with severe disease presented higher hemolysis and higher plasma concentrations of Cu/Zn superoxide dismutase (SOD-1) and lower concentrations of PGE(2) and TGF-beta than those with mild disease. In addition, there was a positive correlation between SOD-1 concentrations and plasma levels of TNF-alpha. During antimalaria treatment, the concentrations of plasma SOD-1 reduced whereas PGE(2) and TGF-beta increased in the individuals severely ill. Using an in vitro model with human mononuclear cells, we demonstrated that the heme effect on the impairment of the production of PGE(2) and TGF-beta partially involves heme binding to CD14 and depends on the production of SOD-1. Aside from furthering the current knowledge about the pathogenesis of vivax malaria, the present results may represent a general mechanism for hemolytic diseases and could be useful for future studies of therapeutic approaches. The Journal of Immunology, 2010, 185: 1196-1204.

Population genetic analysis of large sequence polymorphisms in Plasmodium falciparum blood-stage antigens

Plasmodium falciparum, the causative agent of human malaria, invades host erythrocytes using several proteins on the surface of the invasive merozoite, which have been proposed as potential vaccine candidates. Members of the multi-gene PfRh family are surface antigens that have been shown to play a central role in directing merozoites to alternative erythrocyte receptors for invasion. Recently, we identified a large structural polymorphism, a 0.58 Kb deletion, in the C-terminal region of the PfRh2b gene, present at a high frequency in parasite populations from Senegal. We hypothesize that this region is a target of humoral immunity. Here, by analyzing 371 P. falciparum isolates we show that this major allele is present at varying frequencies in different populations within Senegal, Africa, and throughout the world. For allelic dimorphisms in the asexual stage antigens, Msp-2 and EBA-175, we find minimal geographic differentiation among parasite populations from Senegal and other African localities, suggesting extensive gene flow among these populations and/or immune-mediated frequency-dependent balancing selection. In contrast, we observe a higher level of inter-population divergence (as measured by F(st)) for the PfRh2b deletion, similar to that observed for SNPs from the sexual stage Pfs45/48 loci, which is postulated to be under directional selection. We confirm that the region containing the PfRh2b polymorphism is a target of humoral immune responses by demonstrating antibody reactivity of endemic sera. Our analysis of inter-population divergence suggests that in contrast to the large allelic dimorphisms in EBA-175 and Msp-2, the presence or absence of the large PfRh2b deletion may not elicit frequency-dependent immune selection, but may be under positive immune selection, having important implications for the development of these proteins as vaccine candidates. (C) 2009 Elsevier B.V. All rights reserved.

CD8(+) T cell adjuvant effects of Salmonella FliCd flagellin in live vaccine vectors or as purified protein

Salmonella flagellin, the flagellum structural subunit, has received particular interest as a vaccine adjuvant conferring enhanced immunogenity to soluble proteins or peptides, both for activation of antibody and cellular immune responses. In the present study, we evaluated the Salmonella enterica FliCd flagellin as a T cell vaccine adjuvant using as model the 9-mer (SYVPSAEQI) synthetic H2(d)-restricted CD8(+) T cell-specific epitope (CS(280-288)) derived from the Plasmodium yoelii circumsporozoite (G) protein. The FliCd adjuvant effects were determined under two different conditions: (i) as recombinant flagella, expressed by orally delivered live S. Dublin vaccine strains expressing the target CS(280-288) peptide fused at the central hypervariable domain, and (ii) as purified protein in acellular vaccines in which flagellin was administered to mice either as a recombinant protein fused or admixed with the target CS(280-288) peptide. The results showed that CS(280-288)-specific cytotoxic CD8(+) T cells were primed when BALB/c mice were orally inoculated with the expressing the CS280-288 epitope S. Dublin vaccine strain. In contrast, mice immunized with purified FliCd admixed with the CS280-288 peptide and, to a lesser extent, fused with the target peptide developed specific cytotoxic CD8(+) T cell responses without the need of a heterologous booster immunization. The CD8(+) T cell adjuvant effects of flagellin, either fused or not with the target peptide, correlated with the in vivo activation of CD11c(+) dendritic cells. Taken together, the present results demonstrate that Salmonella flagellins are flexible adjuvant and induce adaptative immune responses when administered by different routes or vaccine formulations. (C) 2009 Elsevier Ltd. All rights reserved.