Variable disruption of epithelial monolayers by Neisseria meningitidis carriage isolates of the hypervirulent MenW cc11 and MenY cc23 lineages.

Colonization of mucosal tissues by Neisseria meningitidis requires adhesion mediated by the type IV pilus and multiple outer-membrane proteins. Penetration of the mucosa and invasion of epithelial cells are thought to contribute to host persistence and invasive disease. Using Calu-3 cell monolayers grown at an air-liquid interface, we examined adhesion, invasion and monolayer disruption by carriage isolates of two clonal complexes of N. meningitidis. Carriage isolates of both the serogroup Y cc23 and the hypervirulent serogroup W cc11 lineages exhibited high levels of cellular adhesion, and a variable disruption phenotype across independent isolates. Inactivation of the gene encoding the main pilus sub-unit in multiple cc11 isolates abrogated both adhesive capacity and ability to disrupt epithelial monolayers. Contrastingly, inactivation of the phase-variable opa or nadA genes reduced adhesion and invasion, but not disruption of monolayer integrity. Adherence of tissue-disruptive meningococci correlated with loss of staining for the tight junction protein, occludin. Intriguingly, in a pilus-negative strain background, we observed compensatory ON switching of opa genes, which facilitated continued adhesion. We conclude that disruption of epithelial monolayers occurs in multiple meningococcal lineages but can vary during carriage and is intimately linked to pilus-mediated adhesion.

Sulfation at Glycopolymer Side Chains Switches Activity at the Macrophage Mannose Receptor (CD206) In Vitro and In Vivo.

The mannose receptor (CD206) is an endocytic receptor expressed by selected innate immune cells and nonvascular endothelium, which plays a critical role in both homeostasis and pathogen recognition. Although its involvement in the development of several diseases and viral infections is well established, molecular tools able to both provide insight on the chemistry of CD206-ligand interactions and, importantly, effectively modulate its activity are currently lacking. Using novel SO4-3-Gal-glycopolymers targeting its cysteine-rich lectin ectodomain, this study uncovers and elucidates a previously unknown mechanism of CD206 blockade involving the formation of stable intracellular SO4-3-Gal-glycopolymer-CD206 complexes that prevents receptor recycling to the cell membrane. Further, we show that SO4-3-Gal glycopolymers inhibit CD206 both in vitro and in vivo, revealing hitherto unknown receptor function and demonstrating their potential as CD206 modulators within future immunotherapies.

C-type lectin receptors MR and DC-SIGN are involved in recognition of hemocyanins, shaping their immunostimulatory effects on human dendritic cells.

Hemocyanins are used as immunomodulators in clinical applications because they induce a strong Th1-biased cell-mediated immunity, which has beneficial effects. They are multiligand glycosylated molecules with abundant and complex mannose-rich structures. It remains unclear whether these structures influence hemocyanin-induced immunostimulatory processes in human APCs. We have previously shown that hemocyanin glycans from Concholepas concholepas (CCH), Fissurella latimarginata (FLH), and Megathura crenulata (KLH), participate in their immune recognition and immunogenicity in mice, interacting with murine C-type lectin receptors (CLRs). Here, we studied the interactions of these hemocyanins with two major mannose-binding CLRs on monocyte-derived human DCs: MR (mannose receptor) and DC-SIGN (DC-specific ICAM-3-grabbing nonintegrin). Diverse analyses showed that hemocyanins are internalized by a mannose-sensitive mechanism. This process was calcium dependent. Moreover, hemocyanins colocalized with MR and DC-SIGN, and were partly internalized through clathrin-mediated endocytosis. The hemocyanin-mediated proinflammatory cytokine response was impaired when using deglycosylated FLH and KLH compared to CCH. We further showed that hemocyanins bind to human MR and DC-SIGN in a carbohydrate-dependent manner with affinity constants in the physiological concentration range. Overall, we showed that these three clinically valuable hemocyanins interact with human mannose-sensitive CLRs, initiating an immune response and promoting a Th1 cell-driving potential.

Capture of influenza by medullary dendritic cells via SIGN-R1 is essential for humoral immunity in draining lymph nodes.

A major pathway for B cell acquisition of lymph-borne particulate antigens relies on antigen capture by subcapsular sinus macrophages of the lymph node. Here we tested whether this mechanism is also important for humoral immunity to inactivated influenza virus. By multiple approaches, including multiphoton intravital imaging, we found that antigen capture by sinus-lining macrophages was important for limiting the systemic spread of virus but not for the generation of influenza-specific humoral immunity. Instead, we found that dendritic cells residing in the lymph node medulla use the lectin receptor SIGN-R1 to capture lymph-borne influenza virus and promote humoral immunity. Thus, our results have important implications for the generation of durable humoral immunity to viral pathogens through vaccination.

Gamma Interferon and Interleukin-17A Differentially Influence the Response of Human Macrophages and Neutrophils to Pseudomonas aeruginosa Infection.

Macrophages are important orchestrators of inflammation during bacterial infection, acting as both effector cells and regulators of neutrophil recruitment and life span. Differently activated macrophage populations with distinct inflammatory and microbicidal potentials have been described. Our previous work unveiled a positive and a negative correlation between levels of gamma interferon (IFN-γ) and interleukin-17A (IL-17A), respectively, and lung function in cystic fibrosis, particularly in patients chronically infected with Pseudomonas aeruginosa This study sought to define key parameters in human antibacterial immunity under Th1- and Th17-dominated inflammatory conditions; the final aim was to identify unique characteristics that could be fine-tuned therapeutically to minimize tissue damage while maximizing bacterial clearance. Toward this aim, neutrophils were incorporated into cultures of macrophages treated with IFN-γ or IL-17A and infected with P. aeruginosa The intent of this design was to model (i) initiation of inflammation by infected macrophages and (ii) delayed arrival of neutrophils and their exposure to macrophage-derived cytokines. Under these conditions, IFN-γ decreased bacterial killing and promoted the production of monocyte chemoattractant protein 1 (MCP-1). In contrast, IL-17A promoted bacterial killing but did not affect MCP-1 production. The level of secretion of the pyrogen IL-1ß was significantly lower in the presence of IFN-γ than in the presence of IL-17A and correlated with levels of the IL1B transcript in infected macrophages. These findings support the validity of this model to investigate human antibacterial immunity. Based on these observations, the protective and damaging roles of IFN-γ and IL-17A, respectively, during P. aeruginosa infection could be caused by their contrasting effects on IL-1ß and MCP-1 production.

Physiological roles of macrophages.

Macrophages are present in mammals from midgestation, contributing to physiologic homeostasis throughout life. Macrophages arise from yolk sac and foetal liver progenitors during embryonic development in the mouse and persist in different organs as heterogeneous, self-renewing tissue-resident populations. Bone marrow-derived blood monocytes are recruited after birth to replenish tissue-resident populations and to meet further demands during inflammation, infection and metabolic perturbations. Macrophages of mixed origin and different locations vary in replication and turnover, but are all active in mRNA and protein synthesis, fulfilling organ-specific and systemic trophic functions, in addition to host defence. In this review, we emphasise selected properties and non-immune functions of tissue macrophages which contribute to physiologic homeostasis.

Antigen targeting reveals splenic CD169+ macrophages as promoters of germinal center B-cell responses.

Ag delivery to specific APCs is an attractive approach in developing strategies for vaccination. CD169(+) macrophages in the marginal zone of the spleen represent a suitable target for delivery of Ag because of their strategic location, which is optimal for the capture of blood-borne Ag and their close proximity to B cells and T cells in the white pulp. Here we show that Ag targeting to CD169(+) macrophages in mice resulted in strong, isotype-switched, high-affinity Ab production and the preferential induction and long-term persistence of Ag-specific GC B cells and follicular Th cells. In agreement with these observations, CD169(+) macrophages retained intact Ag, induced cognate activation of B cells, and increased expression of costimulatory molecules upon activation. In addition, macrophages were required for the production of cytokines that promote B-cell responses. Our results identify CD169(+) macrophages as promoters of high-affinity humoral immune responses and emphasize the value of CD169 as target for Ag delivery to improve vaccine responses.

Immune dysfunction in patients with obstructive jaundice before and after endoscopic retrograde cholangiopancreatography.

This prospective observational study investigated monocyte cytokine responses to lipopolysaccharide (LPS) in patients with obstructive jaundice (OJ) before and after endoscopic biliary drainage. Dendritic cell (DC) subsets and their expression of co-stimulatory molecules were also studied. Forty patients with OJ and ten non-jaundiced patients with normal gastroscopy findings were recruited. Ten healthy volunteers provided control blood samples for immunological assays. Patients with OJ had blood and duodenal mucosa sampled at the time of endoscopic retrograde cholangiopancreatography (ERCP) and further blood sampled during the recovery phase. Monocyte cytokine responses to LPS, DC subsets and co-stimulatory molecule expression were compared with controls. Duodenal morphology and occludin expression were also assessed. Monocytes obtained before ERCP from jaundiced patients demonstrated reduced cytokine responses to endotoxin compared with controls (IL-1ß: 2678 compared with 4631 pg/ml, P=0.04 and IL-6: 3442 compared with 6157 pg/ml, P=0.002). Monocytes from patients with malignancy had poorer responses to endotoxin than from those with benign OJ (IL-1ß: 2025 compared with 3332 pg/ml, P=0.001). After ERCP, the secretion of inflammatory cytokines by monocytes obtained from jaundiced patients increased (IL-1ß: 2150 compared with 2520 pg/ml, P=0.03 and IL-6: 2488 compared with 3250 pg/ml, P=0.01). Occludin expression (85 compared with 95%, P=0.004) and mean duodenal villus height (334 compared with 404 µm, P=0.03) were lower in jaundiced patients. Before biliary drainage, patients with OJ had a higher percentage of myeloid dendritic cells (mDCs) and greater mDC expression of CD40 (P=0.04) and CD86 (P=0.04). Monocytes from patients with OJ had lower proinflammatory cytokine secretion in response to LPS, an effect reversed following biliary drainage.

Nuclear trafficking, histone cleavage and induction of apoptosis by the meningococcal App and MspA autotransporters.

Neisseria meningitidis, a major cause of bacterial meningitis and septicaemia, secretes multiple virulence factors, including the adhesion and penetration protein (App) and meningococcal serine protease A (MspA). Both are conserved, immunogenic, type Va autotransporters harbouring S6-family serine endopeptidase domains. Previous work suggested that both could mediate adherence to human cells, but their precise contribution to meningococcal pathogenesis was unclear. Here, we confirm that App and MspA are in vivo virulence factors since human CD46-expressing transgenic mice infected with meningococcal mutants lacking App, MspA or both had improved survival rates compared with mice infected with wild type. Confocal imaging showed that App and MspA were internalized by human cells and trafficked to the nucleus. Cross-linking and enzyme-linked immuno assay (ELISA) confirmed that mannose receptor (MR), transferrin receptor 1 (TfR1) and histones interact with MspA and App. Dendritic cell (DC) uptake could be blocked using mannan and transferrin, the specific physiological ligands for MR and TfR1, whereas in vitro clipping assays confirmed the ability of both proteins to proteolytically cleave the core histone H3. Finally, we show that App and MspA induce a dose-dependent increase in DC death via caspase-dependent apoptosis. Our data provide novel insights into the roles of App and MspA in meningococcal infection.

CD169+ macrophages at the crossroads of antigen presentation.

CD169(+) macrophages have fascinated immunologists because of their unique distribution in secondary lymphoid organs, redistribution upon immune activation and, lately, because of their contribution to antigen handling. Their association with B cell follicles prompted early studies on their involvement in B cell activation, and recent work has unveiled an unexpected role in facilitating activation of other lymphocyte subsets, such as invariant natural killer T (iNKT) cells. New data also argue that CD169(+) macrophages activate CD8 T cells in response to dead cell-associated antigens in lymph nodes and by transferring antigen to dendritic cells (DCs) in the spleen. Understanding the role of CD169(+) macrophages in the activation of acquired immunity could benefit the design of vaccination strategies, for example those aimed at eliciting cytotoxic T cells.

The macrophage mannose receptor promotes uptake of ADAMTS13 by dendritic cells.

ADAMTS13 is a plasma metalloproteinase that regulates platelet adhesion and aggregation by cleaving ultra-large VWF multimers on the surfaces of endothelial cells. Autoantibodies directed against ADAMTS13 prohibit the processing of VWF multimers, initiating a rare and life-threatening disorder called acquired thrombotic thrombocytopenic purpura. The formation of autoantibodies depends on the activation of CD4(+) T cells. This process requires immune recognition, endocytosis, and subsequent processing of ADAMTS13 into peptides that are presented on MHC class II molecules to CD4(+) T cells by dendritic cells (DCs). In the present study, we investigated endocytosis of recombinant ADAMTS13 by immature monocyte-derived DCs using flow cytometry and confocal microscopy. After incubation of fluorescently labeled ADAMTS13 with DCs, significant uptake of ADAMTS13 was observed. Endocytosis of ADAMTS13 was completely blocked by the addition of EGTA and mannan. ADAMTS13 endocytosis was decreased in the presence of a blocking mAb directed toward the macrophage mannose receptor (MR). Furthermore, siRNA silencing of MR reduced the uptake of ADAMTS13 by DCs. In addition, in vitro binding studies confirmed the interaction of ADAMTS13 with the carbohydrate recognition domains of MR. The results of the present study indicate that sugar moieties on ADAMTS13 interact with MR, thereby promoting its endocytosis by APCs.

Development of a Polymicrobial Colony Biofilm Model to Test Antimicrobials in Cystic Fibrosis.

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as colony-forming units or metabolic activity. Furthermore, many biofilm models do not reflect the biological and physiochemical properties of the human host environment. This is an important issue in many conditions, but most noticeably in cystic fibrosis (CF). A large proportion of people with CF suffer from both chronic and intermittent infections, and in vitro, antibiotic susceptibility testing poorly correlates with patient treatment outcomes. Some biofilm models incorporate CF lung-relevant media, including synthetic sputum mimics, but do not consider the polymicrobial nature of the environment, which alters biofilm architecture, physiology, and the way microbes respond to treatment. The solid-air interface colony biofilm model described here is highly adaptable and incorporates both CF-relevant media and a polymicrobial context. This model can also be used for mid-throughput screening of antimicrobials and to study their effect on polymicrobial dynamics. Output measurements from the model can be colony-forming units, metabolic activity, and confocal microscopy analysis. The model can easily be adapted to different microorganisms, media, temperatures, and variable oxygen conditions and can be used to test a wide range of chemical, biological, and physical treatments.

Charge neutralized poly(ß-amino ester) polyplex nanoparticles for delivery of self-amplifying RNA.

Therapeutic self-amplifying RNA (saRNA) is a promising approach for disease treatment, as it can be administered in lower doses than messenger RNA (mRNA) to achieve comparable protein production levels. However, saRNA requires an appropriate delivery vehicle to protect it during transit and facilitate its transfection. A widely-adopted approach has been to use polycations to condense these large anionic macromolecules into polyplex nanoparticles, however their high charge density often elicits cytotoxic effects. In this study we postulated that we could improve the potency and tolerability of such delivery vehicles by co-formulating poly(ß-amino ester)s saRNA polyplexes with a non-toxic anionic polymer, γ-polyglutamic acid (γ-PGA) to neutralize partially this positive charge. Accordingly, we prepared a poly(ß-amino ester) from 1,6-hexanedioldiacrylate (HDDA) and 4-aminobutanol (ABOL) and initially evaluated the physicochemical properties of the binary polyplexes (i.e. formed from polymer and saRNA only). Optimised binary polyplex formulations were then taken forward for preparation of ternary complexes containing pHDDA-ABOL, saRNA and γ-PGA. Our findings demonstrate that γ-PGA integration into polyplexes significantly enhanced transfection efficacy in HEK293T and A431 cells without affecting polyplex size. Notably, γ-PGA incorporation leads to a pronounced reduction in zeta potential, which reduced the toxicity of the ternary complexes in moDC, NIH3T3, and A431 cells. Furthermore, the presence of γ-PGA contributed to colloidal stability, reducing aggregation of the ternary complexes, as evidenced by insignificant changes in polydispersity index (PDI) after freeze-thaw cycles. Overall, these results suggest that incorporating the appropriate ratio of a polyanion such as γ-PGA with polycations in RNA delivery formulations is a promising way to improve the in vitro delivery of saRNA.

High-throughput phenotype-to-genotype testing of meningococcal carriage and disease isolates detects genetic determinants of disease-relevant phenotypic traits.

Genome-wide association studies (GWAS) with binary or single phenotype data have successfully identified disease-associated genotypes and determinants of antimicrobial resistance. We describe a novel phenotype-to-genotype approach for a major bacterial pathogen that involves simultaneously testing for associations among multiple disease-related phenotypes and linkages between phenotypic variation and genetic determinants. High-throughput assays quantified variation among 163 Neisseria meningitidis serogroup W ST-11 clonal complex isolates for 11 phenotypic traits. A comparison of carriage and two disease subgroups detected significant differences between groups for eight phenotypic traits. Candidate genotypic testing indicated that indels in csw, a capsular biosynthesis gene, were associated with reduced survival in antibody-depleted heat-inactivated serum. GWAS testing detected 341 significant genetic variants (3 single-nucleotide polymorphisms and 338 unitigs) across all traits except serum bactericidal antibody-depleted assays. Growth traits were associated with variants of capsular biosynthesis genes, carbonic anhydrase, and an iron-uptake system while adhesion-linked variation was in pilC2, marR, and mutS. Multiple phase variation states or combinatorial phasotypes were associated with significant differences in multiple phenotypes. Controlling for group effects through regression and recursive random forest approaches detected group-independent effects for nalP with biofilm formation and fetA with a growth trait. Through random forest testing, nine phenotypes were weakly predictive of MenW:cc11 sub-lineage, original or 2013, for disease isolates while three characteristics separated carriage and disease isolates with >80% accuracy. This study demonstrates the power of combining high-throughput phenotypic testing of pathogenically relevant isolate collections with genomics for identifying genetic determinants of specific disease-relevant phenotypes and the pathobiology of microbial pathogens.IMPORTANCENext-generation sequencing technologies have led to the creation of extensive microbial genome sequence databases for several bacterial pathogens. Mining of these databases is now imperative for unlocking the maximum benefits of these resources. We describe a high-throughput methodology for detecting associations between phenotypic variation in multiple disease-relevant traits and a range of genetic determinants for Neisseria meningitidis, a major causative agent of meningitis and septicemia. Phenotypic variation in 11 disease-related traits was determined for 163 isolates of the hypervirulent ST-11 lineage and linked to specific single-nucleotide polymorphisms, short sequence variants, and phase variation states. Application of machine learning algorithms to our data outputs identified combinatorial phenotypic traits and genetic variants predictive of a disease association. This approach overcomes the limitations of generic meta-data, such as disease versus carriage, and provides an avenue to explore the multi-faceted nature of bacterial disease, carriage, and transmissibility traits.

Glycosylation of surface Ig creates a functional bridge between human follicular lymphoma and microenvironmental lectins.

Surface Ig (sIg) of follicular lymphoma (FL) is vital for tumor cell survival. We found previously that the Ig in FL is unusual, because the variable region genes carry sequence motifs for N-glycan addition. These are introduced by somatic mutation and are tumor specific. Unexpectedly, added glycans terminate at high mannose, suggesting a potentially important interaction of FL cells with mannose-binding lectins of the innate immune system. We have now identified mannosylated IgM at the surface of primary lymphoma cells. Recombinant lectin domains of the mannose receptor (MR) or DC-SIGN bind mannosylated Igs in vitro and bind to FL cells, signaling sIgM-associated increases in intracellular Ca(2+). Lectins also bind to normal B cells but fail to signal. In contrast, anti-Ig signaled similarly in both FL and normal B cells. Mannosylation patterns were mimicked by FL Ig-derived single-chain Fvs (scFv), providing probes for potential receptors. Mannosylated scFv bound specifically to the lectin domains of the MR and DC-SIGN and blocked signaling. Mannosylated scFv also bound to DC-SIGN on the surface of dendritic cells. This unique lymphoma-specific interaction of sIg with lectins of innate immunity reveals a potential route for microenvironmental support of tumor cells, mediated via the key B-cell receptor.

Uptake of blood coagulation factor VIII by dendritic cells is mediated via its C1 domain.

BACKGROUND: The uptake and processing of blood coagulation factor VIII (FVIII) by antigen-presenting cells and the subsequent presentation of FVIII-derived peptides to CD4(+) T cells direct the immune response to FVIII in patients with hemophilia A. Multiple receptors including mannose receptor and low-density lipoprotein receptor-related protein-1 (LRP1) have been implicated in FVIII uptake. OBJECTIVE: This work studies the involvement of receptor candidates in FVIII uptake by dendritic cells (DCs). Furthermore, we explore FVIII residues that mediate endocytosis. METHODS: FVIII uptake was performed with human monocyte-derived and murine bone marrow-derived DCs. To investigate FVIII endocytosis, competition assays with soluble receptor ligands, binding studies with recombinant receptor fragments, and small-interfering RNA-induced gene silencing were performed. In addition, FVIII-targeting monoclonal antibodies KM33 and VK34 were used. To confirm in vitro results, hemophilic E17 knockout mice were pretreated with antibodies prior to FVIII injections and anti-FVIII titers were determined. RESULTS: Upon treatment of DCs with mannan or LRP ligand α2-macroglobulin, we observed only a minor decrease in FVIII internalization. In addition, small interfering RNA-mediated knockdown of LRP, mannose receptor, or DC-SIGN expression in monocyte-derived dendritic cells did not prevent FVIII uptake. Binding studies using Fc chimeras revealed that LRP, DC-SIGN, and mannose receptor can bind to FVIII; however, we did not observe a critical role for these receptors in FVIII uptake. Previous studies have shown that human antibodies targeting the C1 (KM33) and A2 (VK34) domains of FVIII interfere with binding to endocytic receptors. Preincubation of FVIII with VK34 did not influence FVIII uptake; however, KM33 completely inhibited FVIII endocytosis by both monocyte-derived dendritic cells and bone marrow-derived dendritic cells. Accordingly, anti-FVIII antibody titers were greatly reduced following the preadministration of KM33 in vivo. CONCLUSION: Together, our observations emphasize the physiological significance of KM33-targeted residues within the C1 domain in the uptake of FVIII by DCs in vitro and in vivo.

Recognition of the major cat allergen Fel d 1 through the cysteine-rich domain of the mannose receptor determines its allergenicity.

Dendritic cells are professional antigen-presenting cells that are specialized in antigen uptake and presentation. Allergy to cat has increased substantially in recent years and has been shown to be positively associated with asthma. We have recently shown that the mannose receptor (MR), a C-type lectin expressed by dendritic cells, recognizes various glycoallergens from diverse sources and is involved in promoting allergic responses to a major house dust mite allergen in vitro. Here we investigated the potential role of MR in allergic responses to Fel d 1, a major cat allergen. Fel d 1 binding to MR was confirmed by ELISA. Using blocking, gene silencing (siRNA) experiments, and MR knock-out (MR(-/-)) cells, we have demonstrated that MR plays a major role in internalization of Fel d 1 by human and mouse antigen-presenting cells. Intriguingly, unlike other glycoallergens, recognition of Fel d 1 by MR is mediated by the cysteine-rich domain, which correlates with the presence of sulfated carbohydrates in natural Fel d 1. WT and MR(-/-) mice were used to study the role of MR in allergic sensitization to Fel d 1 in vivo. MR(-/-) mice sensitized with cat dander extract and Fel d 1 produced significantly lower levels of total IgE, Fel d 1-specific-IgE and IgG1, the hallmarks of allergic response, compared with WT mice. Our data show for the first time that Fel d 1 is a novel ligand of the cysteine-rich domain of MR and that MR is likely to play a pivotal role in allergic sensitization to airborne allergens in vivo.

Fungal recognition enhances mannose receptor shedding through dectin-1 engagement.

The mannose receptor (MR) is an endocytic type I membrane molecule with a broad ligand specificity that is involved in both hemostasis and pathogen recognition. Membrane-anchored MR is cleaved by a metalloproteinase into functional soluble MR (sMR) composed of the extracellular domains of intact MR. Although sMR production was initially considered a constitutive process, enhanced MR shedding has been observed in response to the fungal pathogen Pneumocystis carinii. In this work, we have investigated the mechanism mediating enhanced MR shedding in response to fungi. We show that other fungal species, including Candida albicans and Aspergillus fumigatus, together with zymosan, a preparation of the cell wall of Saccharomyces cerevisiae, mimic the effect of P. carinii on sMR production and that this effect takes place mainly through ß-glucan recognition. Additionally, we demonstrate that MR cleavage in response to C. albicans and bioactive particulate ß-glucan requires expression of dectin-1. Our data, obtained using specific inhibitors, are consistent with the canonical Syk-mediated pathway triggered by dectin-1 being mainly responsible for inducing MR shedding, with Raf-1 being partially involved. As in the case of steady-state conditions, MR shedding in response to C. albicans and ß-glucan particles requires metalloprotease activity. The induction of MR shedding by dectin-1 has clear implications for the role of MR in fungal recognition, as sMR was previously shown to retain the ability to bind fungal pathogens and can interact with numerous host molecules, including lysosomal hydrolases. Thus, MR cleavage could also impact on the magnitude of inflammation during fungal infection.

The mannose receptor mediates the uptake of diverse native allergens by dendritic cells and determines allergen-induced T cell polarization through modulation of IDO activity.

The mannose receptor (MR) is a C-type lectin expressed by dendritic cells (DCs). We have investigated the ability of MR to recognize glycosylated allergens. Using a gene silencing strategy, we have specifically inhibited the expression of MR on human monocyte-derived DCs. We show that MR mediates internalization of diverse allergens from mite (Der p 1 and Der p 2), dog (Can f 1), cockroach (Bla g 2), and peanut (Ara h 1) through their carbohydrate moieties. All of these allergens bind to the C-type lectin-like carbohydrate recognition domains 4-7 of MR. We have also assessed the contribution of MR to T cell polarization after allergen exposure. We show that silencing MR expression on monocyte-derived DCs reverses the Th2 cell polarization bias, driven by Der p 1 allergen exposure, through upregulation of IDO activity. In conclusion, our work demonstrates a major role for MR in glycoallergen recognition and in the development of Th2 responses.

Diurnal Differences in Intracellular Replication Within Splenic Macrophages Correlates With the Outcome of Pneumococcal Infection.

Circadian rhythms affect the progression and severity of bacterial infections including those caused by Streptococcus pneumoniae, but the mechanisms responsible for this phenomenon remain largely elusive. Following advances in our understanding of the role of replication of S. pneumoniae within splenic macrophages, we sought to investigate whether events within the spleen correlate with differential outcomes of invasive pneumococcal infection. Utilising murine invasive pneumococcal disease (IPD) models, here we report that infection during the murine active phase (zeitgeber time 15; 15h after start of light cycle, 3h after start of dark cycle) resulted in significantly faster onset of septicaemia compared to rest phase (zeitgeber time 3; 3h after start of light cycle) infection. This correlated with significantly higher pneumococcal burden within the spleen of active phase-infected mice at early time points compared to rest phase-infected mice. Whole-section confocal microscopy analysis of these spleens revealed that the number of pneumococci is significantly higher exclusively within marginal zone metallophilic macrophages (MMMs) known to allow intracellular pneumococcal replication as a prerequisite step to the onset of septicaemia. Pneumococcal clusters within MMMs were more abundant and increased in size over time in active phase-infected mice compared to those in rest phase-infected mice which decreased in size and were present in a lower percentage of MMMs. This phenomenon preceded significantly higher levels of bacteraemia alongside serum IL-6 and TNF-α concentrations in active phase-infected mice following re-seeding of pneumococci into the blood. These data greatly advance our fundamental knowledge of pneumococcal infection by linking susceptibility to invasive pneumococcal infection to variation in the propensity of MMMs to allow persistence and replication of phagocytosed bacteria. These findings also outline a somewhat rare scenario whereby the active phase of an organism's circadian cycle plays a seemingly counterproductive role in the control of invasive infection.