Enhanced antigen presentation and immunostimulation of dendritic cells using acid-degradable cationic nanoparticles.

Acid-degradable cationic nanoparticles encapsulating a model antigen (i.e., ovalbumin) were prepared by inverse microemulsion polymerization with acid-cleavable acetal cross-linkers. Incubation of these degradable nanoparticles with dendritic cells derived from bone marrow (BMDCs) resulted in the enhanced presentation of ovalbumin-derived peptides, as quantified by B3Z cells, a CD8+ T cell hybridoma. The cationic nature of the particles contributed to the increased surface endocytosis (or phagocytosis) observed with BMDCs, which is the first barrier to overcome for successful antigen delivery. The acid sensitivity of the particles served to direct more ovalbumin antigens to be processed into the appropriately trimmed peptide fragments and presented via the major histocompatibility complex (MHC) class I pathway following hydrolysis within the acidic lysosomes. It was also shown that adjuvant molecules such as unmethylated CpG oligonucleotides (CpG ODN) and anti-interleukin-10 oligonucleotides (AS10 ODN) could be co-delivered with the protein antigen for maximized cellular immune response

IL-10-conditioned dendritic cells, decommissioned for recruitment of adaptive immunity, elicit innate inflammatory gene products in response to danger signals

Dendritic cells (DCs) are the professional APCs of the immune system, enabling T cells to perceive and respond appropriately to potentially dangerous microbes, while also being able to maintain T cell tolerance toward self. In part, such tolerance can be determined by IL-10 released from certain types of regulatory T cells. IL-10 has previously been shown to render DCs unable to activate T cells and it has been assumed that this process represents a general block in maturation. Using serial analysis of gene expression, we show that IL-10 pretreatment of murine bone marrow-derived DCs alone causes significant changes in gene expression. Furthermore, these cells retain the ability to respond to Toll-like receptor agonists, but in a manner skewed toward the selective induction of mediators known to enhance local inflammation and innate immunity, among which we highlight a novel CXCR2 ligand, DC inflammatory protein-1. These data suggest that, while the presence of a protolerogenic and purportedly anti-inflammatory agent such as IL-10 precludes DCs from acquiring their potential as initiators of adaptive immunity, their ability to act as initiators of innate immunity in response to Toll-like receptor signaling is enhanced

Control of RelB during dendritic cell activation integrates canonical and noncanonical NF-κB pathways.

The NF-κB protein RelB controls dendritic cell (DC) maturation and may be targeted therapeutically to manipulate T cell responses in disease. Here we report that RelB promoted DC activation not as the expected RelB-p52 effector of the noncanonical NF-κB pathway, but as a RelB-p50 dimer regulated by canonical IκBs, IκBα and IκBɛ. IκB control of RelB minimized spontaneous maturation but enabled rapid pathogen-responsive maturation. Computational modeling of the NF-κB signaling module identified control points of this unexpected cell type-specific regulation. Fibroblasts that we engineered accordingly showed DC-like RelB control. Canonical pathway control of RelB regulated pathogen-responsive gene expression programs. This work illustrates the potential utility of systems analyses in guiding the development of combination therapeutics for modulating DC-dependent T cell responses

Bone marrow dendritic cells deficient for CD40 and IL-23p19 are tolerogenic in vitro

Objective(s): In addition to pro-inflammatory role, dendritic cells (DCs) can also be anti-inflammatory when they acquire tolerogenic phenotype. In this study we tested the role of CD40 and IL-23p19 in antigen presenting function of bone marrow-derived DCs (BMDCs) by comparing BMDCs derived from CD40 knockout (CD40KO-DCs) and IL-23p19 (IL-23p19KO-DCs) knockout mice with those from C57BL/6 mice (Cont-DCs). We have focused on CD40 and IL-23, as these molecules have well established pro-inflammatory roles in a number of autoimmune and inflammatory diseases. Materials and Methods: The expression of maturation markers MHC II and co-stimulatory molecules CD40, CD80, and CD86 was analyzed by flow cytometry, while the expression of CD40 and IL-23p19 was measured by RT-PCR. The capacity of BMDCs to activate CD4+ T cells was evaluated by 3H-thymidine incorporation, and the capacity of BMDCs to uptake antigen was evaluated using fluorescent OVA and flow cytometry. Results: The lack of CD40 or IL-23p19 had no effect on uptake of FITC-OVA by the DCs, confirming their immature phenotype. Moreover, CD40KO-DCs had significantly reduced capacity to stimulate proliferation of CD4+ T cells. CD4+ T cells activated by CD40KO-DCs and IL-23p19KO-DCs produced significantly less IFN-γ (P-value ≤0.05), while CD4+ T cells stimulated by IL-23p19KO-DCs produced less GM-CSF and more IL-10 than Cont-DCs. Conclusion: This study shows that CD40KO-DCs and IL-23p19KO-DCs have a marked tolerogenic potency in vitro. Future in vivo studies should determine if and to what extent DCs lacking CD40 or IL-23 have a potential to be useful in therapy of autoimmune inflammation

A mucin-like peptide from Fasciola hepatica instructs dendritic cells with parasite specific Th1-polarizing activity

Fasciolosis is a trematode zoonosis of interest in public health and cattle production. We report here the immunostimulatory effect of a 66 mer mucin-like peptide from Fasciola hepatica (Fhmuc), which synergizes with lipopolysaccharide (LPS) to promote dendritic cell (DC) maturation, endowing these cells with Th1-polarizing capacity. Exposure of DCs to Fhmuc in presence of LPS induced enhanced secretion of pro-inflammatory cytokines and expression of co-stimulatory molecules by DCs, promoting their T cell stimulatory capacity and selectively augmenting IFN- secretion by allogeneic T cells. Furthermore, exposure of DCs to Fhmuc augmented LPS-induced Toll-like receptor (TLR) 4 expression on the cell surface. Finally, Fhmuc-conditioned DCs induced parasite specific-adaptive immunity with increased levels of IFN-gamma secreted by splenocytes from vaccinated animals, and higher parasite-specific IgG antibodies. However, DC-treated Fhmuc conferred modest protection against F. hepatica infection highlighting the potent immuno-regulatory capacity of the parasite. In summary, this work highlights the capacity of a mucin-derived peptide from F. hepatica to enhance LPS-maturation of DCs and induce parasite-specific immune responses with potential implications in vaccination and therapeutic strategies.Fil: Noya, Verónica. Universidad de la República; UruguayFil: Brossard, Natalie. Universidad de la República; UruguayFil: Rodríguez, Ernesto. Universidad de la República; UruguayFil: Dergan Dylon, Leonardo Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Carmona, Carlos. Universidad de la República; UruguayFil: Rabinovich, Gabriel Adrián. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Freire, Teresa. Universidad de la República; Urugua

Direct Inhibition of T-Cell Responses by the Cryptococcus Capsular Polysaccharide Glucuronoxylomannan

The major virulence factor of the pathogenic fungi Cryptococcus neoformans and C. gattii is the capsule. Glucuronoxylomannan (GXM), the major component of the capsule, is a high-molecular-weight polysaccharide that is shed during cryptococcosis and can persist in patients after successful antifungal therapy. Due to the importance of T cells in the anticryptococcal response, we studied the effect of GXM on the ability of dendritic cells (DCs) to initiate a T-cell response. GXM inhibited the activation of cryptococcal mannoprotein-specific hybridoma T cells and the proliferation of OVA-specific OT-II T cells when murine bone marrow-derived DCs were used as antigen-presenting cells. Inhibition of OT-II T-cell proliferation was observed when either OVA protein or OVA323-339 peptide was used as antigen, indicating GXM did not merely prevent antigen uptake or processing. We found that DCs internalize GXM progressively over time; however, the suppressive effect did not require DCs, as GXM directly inhibited T-cell proliferation induced by anti-CD3 antibody, concanavalin A, or phorbol-12-myristate-13-acetate/ionomycin. Analysis of T-cell viability revealed that the reduced proliferation in the presence of GXM was not the result of increased cell death. GXM isolated from each of the four major cryptococcal serotypes inhibited the proliferation of human peripheral blood mononuclear cells stimulated with tetanus toxoid. Thus, we have defined a new mechanism by which GXM can impart virulence: direct inhibition of T-cell proliferation. In patients with cryptococcosis, this could impair optimal cell-mediated immune responses, thereby contributing to the persistence of cryptococcal infections. SynopsisInfections due to the pathogenic yeast Cryptococcus are a significant cause of morbidity and mortality in persons with impaired T-cell functions, particularly those with AIDS. The major virulence factor of Cryptococcus is its capsule, which is composed primarily of the polysaccharide glucuronoxylomannan (GXM). The capsule not only surrounds the organism but also is shed during cryptococcosis. GXM is taken up by macrophages in vitro and in vivo; however, little is known about the interaction between GXM and dendritic cells, which are the most potent cells capable of activating T cells. Because of the importance of T cells in the anticryptococcal response, the authors investigated the effect of GXM on the ability of dendritic cells to initiate a T-cell response. They found the polysaccharide was internalized by dendritic cells and inhibited antigen-specific T-cell responses. Furthermore, GXM had a direct, inhibitory effect on T-cell proliferation, independent of the effect on dendritic cells. These findings may help explain the persistence of cryptococcal infections and suggest that GXM could be therapeutic in situations where suppression of T-cell responses is desired.National Institutes of Health (R01 AI25780, R01 AI066087, R01 AI37532

Increased S-nitrosylation and proteasomal degradation of caspase-3 during infection contribute to the persistence of adherent invasive escherichia coli (AIEC) in immune cells

Adherent invasive Escherichia coli (AIEC) have been implicated as a causative agent of Crohn's disease (CD) due to their isolation from the intestines of CD sufferers and their ability to persist in macrophages inducing granulomas. The rapid intracellular multiplication of AIEC sets it apart from other enteric pathogens such as Salmonella Typhimurium which after limited replication induce programmed cell death (PCD). Understanding the response of infected cells to the increased AIEC bacterial load and associated metabolic stress may offer insights into AIEC pathogenesis and its association with CD. Here we show that AIEC persistence within macrophages and dendritic cells is facilitated by increased proteasomal degradation of caspase-3. In addition S-nitrosylation of pro- and active forms of caspase-3, which can inhibit the enzymes activity, is increased in AIEC infected macrophages. This S-nitrosylated caspase-3 was seen to accumulate upon inhibition of the proteasome indicating an additional role for S-nitrosylation in inducing caspase-3 degradation in a manner independent of ubiquitination. In addition to the autophagic genetic defects that are linked to CD, this delay in apoptosis mediated in AIEC infected cells through increased degradation of caspase-3, may be an essential factor in its prolonged persistence in CD patients

Glycans from Fasciola hepatica modulate the host immune response and TLR-Induced maturation of dendritic cells

Helminths express various carbohydrate-containing glycoconjugates on their surface, and they release glycan-rich excretion/secretion products that can be very important in their life cycles, infection and pathology. Recent evidence suggests that parasite glycoconjugates could play a role in the evasion of the immune response, leading to a modified Th2-polarized immune response that favors parasite survival in the host. Nevertheless, there is limited information about the nature or function of glycans produced by the trematode Fasciola hepatica, the causative agent of fasciolosis. In this paper, we investigate whether glycosylated molecules from F. hepatica participate in the modulation of host immunity. We also focus on dendritic cells, since they are an important target of immune-modulation by helminths, affecting their activity or function. Our results indicate that glycans from F. hepatica promote the production of IL-4 and IL-10, suppressing IFNγ production. During infection, this parasite is able to induce a semi-mature phenotype of DCs expressing low levels of MHCII and secrete IL-10. Furthermore, we show that parasite glycoconjugates mediate the modulation of LPS-induced maturation of DCs since their oxidation restores the capacity of LPS-treated DCs to secrete high levels of the pro-inflammatory cytokines IL-6 and IL-12/23p40 and low levels of the anti-inflammatory cytokine IL-10. Inhibition assays using carbohydrates suggest that the immune-modulation is mediated, at least in part, by the recognition of a mannose specific-CLR that signals by recruiting the phosphatase Php2. The results presented here contribute to the understanding of the role of parasite glycosylated molecules in the modulation of the host immunity and might be useful in the design of vaccines against fasciolosis.Fil: Rodriguez, Ernesto. Universidad de la República; UruguayFil: Noya, Verónica. Universidad de la República; UruguayFil: Cervi, Laura Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Chiribao, Maria Laura. Universidad de la República; UruguayFil: Brossard, Natalie. Universidad de la República; UruguayFil: Chiale, Carolina. Universidad de la República; UruguayFil: Carmona, Carlos. Universidad de la República; UruguayFil: Giacomini, Cecilia. Universidad de la República; UruguayFil: Freire, Teresa. Universidad de la República; Urugua

MARCH1 protects the lipid raft and tetraspanin web from MHCII proteotoxicity in dendritic cells

Dendritic cells (DCs) produce major histocompatibility complex II (MHCII) in large amounts to function as professional antigen presenting cells. Paradoxically, DCs also ubiquitinate and degrade MHCII in a constitutive manner. Mice deficient in the MHCII-ubiquitinating enzyme membrane-anchored RING-CH1, or the ubiquitin-acceptor lysine of MHCII, exhibit a substantial reduction in the number of regulatory T (Treg) cells, but the underlying mechanism was unclear. Here we report that ubiquitin-dependent MHCII turnover is critical to maintain homeostasis of lipid rafts and the tetraspanin web in DCs. Lack of MHCII ubiquitination results in the accumulation of excessive quantities of MHCII in the plasma membrane, and the resulting disruption to lipid rafts and the tetraspanin web leads to significant impairment in the ability of DCs to engage and activate thymocytes for Treg cell differentiation. Thus, ubiquitin-dependent MHCII turnover represents a novel quality-control mechanism by which DCs maintain homeostasis of membrane domains that support DC's Treg cell-selecting function