Murine pattern recognition receptor dectin-1 is essential in the development of experimental autoimmune uveoretinitis.

Mycobacteria in complete Freund's adjuvant (CFA) are an essential component of immunization protocols in a number of autoimmune disease animal models including experimental autoimmune encephalomyelitis and uveoretinitis (EAE and EAU, respectively). We determined the role in EAU of two C-type lectin receptors on myeloid cells that recognize and respond to mycobacteria. Using receptor-specific antibodies and knockout mice, we demonstrated for the first time that the macrophage mannose receptor delays disease development but does not affect severity. In contrast, dectin-1 is critically involved in the development of CFA-mediated EAU. Disease severity is reduced in dectin-1 knockout mice and antibody blockade of dectin-1 during the induction, but not the effector phase, prevents EAU development. Significantly, similar blockade of dectin-1 in vivo has no effect in non-CFA-mediated, spontaneously induced or adoptive transfer models of EAU. Thus dectin-1 plays a critical role in the ability of complete Freund's adjuvant to induce EAU in mice.

Characterisation of tumour-infiltrating macrophages: impact on response and survival in patients receiving primary chemotherapy for breast cancer.

The role of the tumour microenvironment and complex cellular interactions has attracted interest in responses to primary chemotherapy. Of particular interest are tumour-infiltrating T cells and tumour-infiltrating macrophages (TIMs). We evaluated TIMs and their key activation markers in patients with breast cancer undergoing primary chemotherapy related to response and survival. One hundred and ninety nine patients with large or locally advanced breast cancers received primary chemotherapy. Clinical data, histopathological responses to chemotherapy and survival were examined related to infiltrating cells in tumour microenvironments: cluster of differentiation (CD)3 (pan T cell); CD4 (helper T cells); CD8 (cytotoxic T cells); CD25 (activated T cells); CD68, suppressor of cytokine signalling (SOCS)1, SOCS3 (macrophages); and CD11c and CD205 (dendritic). In tumours demonstrating better responses to chemotherapy, there were significantly fewer CD4(+) T-helper cells than a poorer response (p < 0.05). There were increased numbers of SOCS3 expressing macrophages (pro-inflammatory) in tumours with complete pathological responses compared with no response to chemotherapy (p < 0.05). There was no association between SOCS1 expressing macrophages (anti-inflammatory) and tumour response. Multivariate analysis revealed that factors indicating better survival were receiving anthracycline plus docetaxel (ExpB = 1.166; p = 0.006), better pathological chemotherapy response (ExpB = 0.309; p = 0.009) and a low macrophage SOCS1 expression (ExpB = 13.465; p = 0.044). This study highlights the heterogeneity of TIMs and provides further insight into complex interactions within tumours. The results emphasise the importance of characterising activation status of infiltrating macrophages and provides proof of principle for using macrophage SOCS protein expression as a survival predictor. The apparent impact of macrophage subsets on overall survival underlines the therapeutic potential of manipulating macrophage activation in cancer.

Murine bone marrow-derived dendritic cells and T-cell activation by Candida albicans.

Dendritic cells (DCs) detect and respond to microbes or their components by producing cytokines and other molecules that can activate the proliferation and differentiation pathways of T cells. Investigation of DC responses to pathogens would thus provide important insights into how T-cell responses most appropriate for the pathogen are induced. Here, we describe methods for the use of mixed leukocyte reactions, to determine the proliferative and cytokine responses of murine splenic T cells in response to co-culture with bone marrow-derived DCs stimulated with Candida albicans.

The contribution of naturally occurring IgM antibodies, IgM cross-reactivity and complement dependency in murine humoral responses to pneumococcal capsular polysaccharides.

Immunogenicity of 12 capsular polysaccharides (CPS) from Streptococcus pneumoniae did not correlate with pre-existing levels of natural IgM anti-CPS antibodies in mice. Immunization of mice with individual CPS, with the exception of type 14 (the only neutral CPS tested), increased serum IgM that also bound other CPS serotypes independent of structural similarity or commonly known contaminants. Surprisingly only IgM response to type 4 (which has a small immunodominant epitope) was dependent on either complement C3 or complement receptors CD35/CD21. IgG anti-CPS responses were infrequently induced, but critically dependent on complement. Our results have clarified the role of complement in the induction of IgM and IgG anti-CPS antibody responses in mice and have implications for CPS vaccine development.

Role of lipopolysaccharide in the induction of type I interferon-dependent cross-priming and IL-10 production in mice by meningococcal outer membrane vesicles.

We investigated the contribution of lipopolysaccharide (LPS) to adjuvant properties of native outer membrane vesicles (NOMV), a vaccine candidate for meningococcal B disease. NOMV induce the maturation of and cytokine production by murine bone marrow-derived dendritic cells through both toll-like receptors (TLR) 2 and 4 which are mostly dependent on the signalling adaptor MyD88. NOMV are also able to induce B cell proliferation in splenocytes from LPS-hyporesponsive mice. However, induction of IL-10 and type I interferon-dependent, antigen-specific and IFN(gamma)-secreting CD8(+) cytotoxic T lymphocyte responses in vivo by NOMV requires LPS. The importance of LPS in the induction of IL-10 and functional cross-priming has implications for NOMV-based vaccine and adjuvant development.

The role of beta2 integrins and lipopolysaccharide-binding protein in the phagocytosis of dead Neisseria meningitidis.

Phagocytosis of microbial pathogens is essential for the host immune response to infection. Our previous work has shown that lipooligosaccharide (LOS) expression on the surface of Neisseria meningitidis (Nm) is essential for phagocytosis, but the receptor involved remained unclear. In this study, we show that human CR3 (CD11b/CD18) and CR4 (CD11c/CD18) are phagocytic receptors for Nm as illustrated by the capacity of CR3- and CR4-transfected Chinese hamster ovary (CHO) cells to facilitate Nm uptake. A CR3-signalling mutant failed to internalize Nm, showing that the ability of CR3 to signal is essential for phagocytosis. Internalization of Nm by CR3-transfected CHO cells could be inhibited by the presence of CR3-specific antibodies. Furthermore, dendritic cells from leukocyte adhesion deficiency-1 patients, who have diminished expression of beta2 integrins, showed markedly reduced phagocytosis of Nm. The CR3-mediated phagocytosis required the presence of lipopolysaccharide-binding protein (LBP). Furthermore, the expression of LOS by Nm was essential for LBP binding and phagocytosis via CR3. These results reveal a critical role of CR3 and LBP in the phagocytosis of Nm and provide important insights into the initial interaction meningococci have with the immune system.

Preferential induction of CD4+ T cell responses through in vivo targeting of antigen to dendritic cell-associated C-type lectin-1.

Targeting of Ags and therapeutics to dendritic cells (DCs) has immense potential for immunotherapy and vaccination. Because DCs are heterogeneous, optimal targeting strategies will require knowledge about functional specialization among DC subpopulations and identification of molecules for targeting appropriate DCs. We characterized the expression of a fungal recognition receptor, DC-associated C-type lectin-1 (Dectin-1), on mouse DC subpopulations and investigated the ability of an anti-Dectin-1 Ab to deliver Ag for the stimulation of immune responses. Dectin-1 was shown to be expressed on CD8alpha-CD4-CD11b+ DCs found in spleen and lymph nodes and dermal DCs present in skin and s.c. lymph nodes. Injection of Ag-anti-Dectin-1 conjugates induced CD4+ and CD8+ T cell and Ab responses at low doses where free Ag failed to elicit a response. Notably, qualitatively different immune responses were generated by targeting Ag to Dectin-1 vs CD205, a molecule expressed on CD8alpha+CD4-CD11b- DCs, dermal DCs, and Langerhans cells. Unlike anti-Dectin-1, anti-CD205 conjugates failed to elicit an Ab response. Moreover, when conjugates were injected i.v., anti-Dectin-1 stimulated a much stronger CD4+ T cell response and a much weaker CD8+ T cell response than anti-CD205. The results reveal Dectin-1 as a potential targeting molecule for immunization and have implications for the specialization of DC subpopulations.

Human MICL (CLEC12A) is differentially glycosylated and is down-regulated following cellular activation.

C-type lectins are the most diverse and prevalent lectin family in immunity. Particular interest has recently been attracted by the C-type lectin-like receptors on NK cells, which appear to regulate the activation/inhibitory balance of these cells, controlling cytotoxicity and cytokine production. We previously identified a human C-type lectin-like receptor, closely related to both the beta-glucan receptor and the lectin-like receptor for oxidized-LDL, named MICL (myeloid inhibitory C-type lectin-like receptor), which we had shown using chimeric analysis to function as an inhibitory receptor. Using a novel MICL-specific monoclonal antibody, we show here that human MICL is expressed primarily on myeloid cells, including granulocytes, monocytes, macrophages, and dendritic cells. Although MICL was highly N-glycosylated in primary cells, the level of glycosylation was found to vary between cell types. MICL surface expression was down-regulated during inflammatory/activation conditions in vitro, as well as during an in vivo model of acute inflammation, which we characterize here. This suggests that human MICL may be involved in the control of myeloid cell activation during inflammation.

IFN-alpha/beta-dependent cross-priming induced by specific toll-like receptor agonists.

Toll-like receptors (TLR) are pattern recognition receptors that have been identified as crucial in the initiation of innate immune responses against pathogens. They are thought to be involved in shaping appropriate adaptive immune responses, although their precise contribution has not yet been fully characterised. Our aim was to investigate in vivo the effect of different TLR stimuli on cellular immune responses. We examined the ability of a range of TLR stimuli to induce CD8+ T cell responses against a model soluble protein antigen, ovalbumin (OVA). We found that TLR 3, TLR 4, and TLR 9 agonists induced functional cross-priming, and that this process was dependent on IFN-alpha/beta signalling pathway.

Induction of CD8+ T cell responses through targeting of antigen to Dectin-2.

Targeted delivery of antigens to dendritic cells (DC) can be used to optimise immunisation. We investigated whether the efficacy with which immune responses are induced can be improved by targeting Ags to a C-type lectin receptor, Dectin-2. When anti-Dectin-2 mAbs were injected s.c., mAb binding was detected on a low percentage of DC in the draining lymph node. Ag conjugated to anti-Dectin-2 mAbs was presented efficiently to CD8+ T cells in vivo and elicited CD8+ T cell responses at low doses where free Ag failed to induce a response. The results reveal Dectin-2 as a potential targeting molecule for immunisation.

Intranasal immunisation with inactivated RSV and bacterial adjuvants induces mucosal protection and abrogates eosinophilia upon challenge.

We have previously shown that following intranasal exposure to influenza virus, specific plasma cells are generated in the nasal-associated lymphoid tissue (NALT) and maintained for the life of the animal. However, we also showed that following infection with respiratory syncytial virus (RSV), specific plasma cells are generated in the NALT but wane quickly and are not maintained even after challenge, even though RSV-specific serum antibody responses remain robust. Only infection with influenza virus generated sterilising immunity, implying a role for these long-lived plasma cells in protection. We show here that the RSV-specific IgA NALT plasma cell population and lung antibody levels can be substantially boosted, both at acute and memory time points, by intranasal immunisation with inactivated RSV (iRSV) in combination with bacterial outer membrane vesicles (OMV) compared to live RSV alone. Finally, challenge with live RSV showed that immunisation with iRSV and OMV protect against both virus replication in the lung and the eosinophil infiltrate generated by either live RSV or iRSV alone. These data show that immunisation with iRSV and OMV maintains a NALT RSV-specific plasma cell population and generates an efficient protective immune response following RSV infection.

The carbohydrate-recognition domain of Dectin-2 is a C-type lectin with specificity for high mannose.

We examined the carbohydrate-binding potential of the C-type lectin-like receptor Dectin-2 (Clecf4n). The carbohydrate-recognition domain (CRD) of Dectin-2 exhibited cation-dependent mannose/fucose-like lectin activity, with an IC(50) for mannose of approximately 20 mM compared to an IC(50) of 1.5 mM for the macrophage mannose receptor when assayed by similar methodology. The extracellular domain of Dectin-2 exhibited binding to live Candida albicans and the Saccharomyces-derived particle zymosan. This binding was completely abrogated by cation chelation and was competed by yeast mannans. We compared the lectin activity of Dectin-2 with that of two other C-type lectin receptors (mannose receptor and SIGNR1) known to bind fungal mannans. Both mannose receptor and SIGNR1 were able to bind bacterial capsular polysaccharides derived from Streptococcus pneumoniae, but interestingly they exhibited distinct binding profiles. The Dectin-2 CRD exhibited only weak interactions to some of these capsular polysaccharides, indicative of different structural or affinity requirements for binding, when compared with the other two lectins. Glycan array analysis of the carbohydrate recognition by Dectin-2 indicated specific recognition of high-mannose structures (Man(9)GlcNAc(2)). The differences in the specificity of these three mannose-specific lectins indicate that mannose recognition is mediated by distinct receptors, with unique specificity, that are expressed by discrete subpopulations of cells, and this further highlights the complex nature of carbohydrate recognition by immune cells.

Dectin-2 is predominantly myeloid restricted and exhibits unique activation-dependent expression on maturing inflammatory monocytes elicited in vivo.

Dectin-2 is a recently described dendritic-cell-associated receptor, suggested to be involved in the initiation and maintenance of UV-induced tolerance. To understand the physiological relevance of the proposed functions of this C-type lectin-like receptor, we have generated monoclonal antibodies against its extracellular domain and performed a detailed study of its expression. In naive mice, Dectin-2 has a novel distribution pattern compared with other myeloid markers, but is predominantly expressed by a wide variety of tissue macrophages. Its expression was limited on dendritic cells and notably absent from brain microglia and choroid plexus or meningeal macrophages. On peripheral blood monocytes, Dectin-2 expression was very low on the surface but was transiently and markedly up-regulated on induction of inflammation in vivo using a variety of stimuli. This change in Dectin-2 expression occurs on 'inflammatory' monocytes after arrival at the inflammatory lesion as demonstrated by adoptive cell-transfer studies, and is independent of whether the macrophages elicited by the stimuli ultimately expressed Dectin-2. These observations show Dectin-2 expression to be characteristic of monocyte activation/maturation at an inflammatory lesion and provide a new perspective on the interpretation of Dectin-2 function in vivo.

The human beta-glucan receptor is widely expressed and functionally equivalent to murine Dectin-1 on primary cells.

We identified the C-type-lectin-like receptor, Dectin-1, as the major receptor for fungal beta-glucans on murine macrophages and have demonstrated that it plays a significant role in the cellular response to these carbohydrates. Using two novel, isoform-specific mAb, we show here that human Dectin-1, the beta-glucan receptor (betaGR), is widely expressed and present on all monocyte populations as well as macrophages, DC, neutrophils and eosinophils. This receptor is also expressed on B cells and a subpopulation of T cells, demonstrating that human Dectin-1 is not myeloid restricted. Both major functional betaGR isoforms - betaGR-A and betaGR-B - were expressed by these cell populations in peripheral blood; however, only betaGR-B was significantly expressed on mature monocyte-derived macrophages and immature DC, suggesting cell-specific control of isoform expression. Inflammatory cells, recruited in vivo using a new skin-window technique, demonstrated that Dectin-1 expression was not significantly modulated on macrophages during inflammation, but is decreased on recruited granulocytes. Despite previous reports detailing the involvement of other beta-glucan receptors on mature human macrophages, we have demonstrated that Dectin-1 acted as the major beta-glucan receptor on these cells and contributed to the inflammatory response to these carbohydrates.

Human dendritic cell activation by Neisseria meningitidis: phagocytosis depends on expression of lipooligosaccharide (LOS) by the bacteria and is required for optimal cytokine production.

Group B Neisseria meningitidis is a human pathogen, for which a universally effective vaccine is still not available. Immune responses to bacteria are initiated by dendritic cells (DC), which internalize and process bacterial antigens for presentation to T cells. We show here that optimal IL-12 and TNF-alpha production by human monocyte derived DC in response to killed serogroup B N. meningitidis depends on physical contact and internalization of the bacteria by DC. The majority of DC producing cytokines had internalized N. meningitidis while inhibition of bacterial internalization markedly impaired IL-12 and TNF-alpha, but not IL-6 production. Internalization of N. meningitidis was shown to depend on lipooligosaccharide (LOS) expressed by the bacteria with poor internalization of LOS deficient bacteria compared to wild-type bacteria. Restoration of LOS biosynthesis in a LOS regulatory strain also restored both internalization and cytokine production and was enhanced in the presence of LPS binding protein (LBP). These results suggest that DC phagocytosis depends on expression of LOS within the bacteria and that optimal cytokine production, particularly IL-12, requires internalization of the bacteria. These findings have important implications for designing vaccines that will induce protective immune responses to group B N. meningitidis.

Expression of the beta-glucan receptor, Dectin-1, on murine leukocytes in situ correlates with its function in pathogen recognition and reveals potential roles in leukocyte interactions.

Dectin-1 is a pathogen-recognition receptor on macrophages (MPhis), neutrophils, and dendritic cells (DCs). On MPhis and bone marrow-derived DCs, it has been shown to mediate the nonopsonic recognition of and response to soluble and particulate yeast beta-glucans. We have optimized the immunohistochemical detection of Dectin-1 and demonstrated its expression on neutrophils, subpopulations of MPhis in splenic red and white pulp, alveolar MPhis, Kupffer cells, and MPhis and DCs in the lamina propria of gut villi. This is consistent with its role in pathogen surveillance. A significant proportion of CD11c(+) splenic DCs expressed Dectin-1, but expression was not restricted to any one subset. Dectin-1 expression was low on resident MPhis and DCs of skin and was not detected on resident MPhis or DCs in kidney, heart, brain, or eye. The proposed, additional role of Dectin-1 as a coreceptor for T cell activation is supported by its expression on DCs in the T cell areas of the spleen and lymph nodes. Strong expression of Dectin-1 on subpopulations of MPhis and DCs in the medullary and corticomedullary regions of the thymus suggests a role distinct from pathogen recognition. Tissue localization thus revealed potential roles of Dectin-1 in leukocyte interactions during innate immune responses and T cell development.

Local and systemic antibody responses in mice immunized intranasally with native and detergent-extracted outer membrane vesicles from Neisseria meningitidis.

The mouse humoral immune response toward native or detergent-extracted outer membrane vesicles (NOMVs and DOMVs, respectively) from Neisseria meningitidis was determined after intranasal immunization. Both preparations elicited high frequencies of NOMV-specific antibody-forming cells (AFCs) locally in the nasal associated lymphoid tissue (NALT) after three or four weekly doses. The diffuse NALT (D-NALT) contained ca. 10-fold more NOMV-specific AFCs than those observed in the mediastinal lymph node, spleen, and bone marrow. AFCs observed in the D-NALT were primarily immunoglobulin A positive (IgA(+)) and were maintained for at least 1 month. In contrast, the organized NALT (O-NALT) contained low numbers of AFCs, and the response was relatively short-lived. In other lymphoid tissues, AFCs producing various IgG subclasses and IgM were present with IgG2b-producing AFCs being dominant or codominant with IgA or IgG2a. In serum and in all of the tissues examined, with the exception of the NALT, NOMVs clearly induced a stronger antibody response and a broader range of antibody isotypes than DOMVs. The development of NOMV-specific AFCs in spleen and bone marrow after intranasal immunization was slow compared to intravenous immunization but, once established, the intranasally elicited responses increased steadily for at least 75 days. NOMV-specific antibodies induced via several routes of immunization had high bactericidal activities in serum. Our results indicated that intranasally administered OMVs induced strong local and systemic antibody responses in mice that were relatively long-lived.

Development of a specific system for targeting protein to metallophilic macrophages.

The cysteine-rich domain (CR) of the mannose receptor binds sulfated glycoprotein CR ligand (CRL) expressed by subpopulations of myeloid cells in secondary lymphoid organs (CRL(+) cells). In naïve mice, these CRL(+) cells, metallophilic macrophages (M) in spleen and subcapsular sinus M in lymph nodes, are located strategically for antigen capture and are adjacent to B cell follicles, but their role in the immune response is unknown. We have exploited the lectin activity of CR to develop a highly specific system for targeting protein to CRL(+) M. We demonstrate that the sulfated carbohydrates recognized by CR are exposed to the extracellular milieu and mediate highly specific targeting of CR-containing proteins. This model will allow the dissection of the role of metallophilic M in an immune response in vivo.

Dectin-1 expression and function are enhanced on alternatively activated and GM-CSF-treated macrophages and are negatively regulated by IL-10, dexamethasone, and lipopolysaccharide.

Dectin-1 is the major macrophage receptor for beta-glucans and generates a proinflammatory response through the recognition of these carbohydrates on fungal pathogens. We have examined the effects of cytokines and other agents on the expression and functions of dectin-1 in both resident and elicited murine peritoneal macrophages (Mphi). Dectin-1 expression was found to be highly up-regulated by GM-CSF and by the cytokines that induce alternative macrophage activation, IL-4 and IL-13. In contrast, IL-10, LPS, and dexamethasone, but not IFN-gamma, down-regulated the expression of this receptor. Modulation of dectin-1 receptor levels correlated with the ability of these macrophages to bind zymosan and significantly affected the contribution of this receptor to the resultant proinflammatory response, as measured by the production of TNF-alpha, although some Mphi-specific differences were observed. These results correlate with the known effects of these cytokines and other agents on the ability of the immune system to recognize and respond to fungal pathogens.

Analysis of mannose receptor regulation by IL-4, IL-10, and proteolytic processing using novel monoclonal antibodies.

The study of the murine macrophage mannose receptor (MR) has been hampered by the lack of specific reagents. We have generated and characterized novel anti-MR monoclonal antibodies and used them to analyze MR expression in primary mouse macrophages (M(phi)). In BioGel- and thioglycollate-elicited M(phi), interleukin (IL)-4 up-regulated total cell-associated MR (cMR), correlating with enhanced surface expression. We investigated the influence of IL-10, a well-characterized deactivator of M(phi) function, on MR levels and observed that it had a similar effect to IL-4. In both cases, enhanced cMR levels translated into increased production of the soluble form of the receptor (sMR). We have demonstrated the presence of sMR in cultures of stable non-M(phi) transductants expressing full-length MR, indicating that the proteolytic activity responsible for cMR cleavage is not M(phi)-restricted. These data support a role for the MR in T helper cell type 2 cytokine-driven, immune responses and suggest a non-M(phi) contribution to sMR production in vivo.