Pattern-recognition receptors (PRRs) detect molecular signatures of microbes and initiate immune responses to infection. Prototypical PRRs such as Toll-like receptors (TLRs) signal via a conserved pathway to induce innate response genes. In contrast, the signaling pathways engaged by other classes of putative PRRs remain ill defined. Here, we demonstrate that the beta-glucan receptor Dectin-1, a yeast binding C type lectin known to synergize with TLR2 to induce TNF alpha and IL-12, can also promote synthesis of IL-2 and IL-10 through phosphorylation of the membrane proximal tyrosine in the cytoplasmic domain and recruitment of Syk kinase. syk-/- dendritic cells (DCs) do not make IL-10 or IL-2 upon yeast stimulation but produce IL-12, indicating that the Dectin-1/Syk and Dectin-1/TLR2 pathways can operate independently. These results identify a novel signaling pathway involved in pattern recognition by C type lectins and suggest a potential role for Syk kinase in regulation of innate immunity.

Toll-like receptors (TLR) recognize microbial and viral patterns and activate dendritic cells (DC). TLR distribution among human DC subsets is heterogeneous: plasmacytoid DC (PDC) express TLR1, 7 and 9, while other DC types do not express TLR9 but express other TLR. Here, we report that mRNA for most TLR is expressed at similar levels by murine splenic DC sub-types, including PDC, but that TLR3 is preferentially expressed by CD8 § + DC while TLR5 and TLR7 are selectively absent from the same subset. Consistent with the latter, TLR7 ligand activates CD8 § -DC and PDC, but not CD8 § + DC as measured by survival ex vivo, upregulation of surface markers and production of IL-12p40. These data suggest that the dichotomy in TLR expression between plasmacytoid and non-plasmacytoid DC is not conserved between species. However, lack of TLR7 expression could restrict the involvement of CD8 § + DC in recognition of certain mouse pathogens.

CD40 ligation triggers IL-12 production by dendritic cells (DC) in vitro. Here, we demonstrate that CD40 cross-linking alone is not sufficient to induce IL-12 production by DC in vivo. Indeed, resting DC make neither the IL-12 p35 nor IL-12 p40 subunits and express only low levels of CD40. Nevertheless, after DC activation by microbial stimuli that primarily upregulate IL-12 p40 and augment CD40 expression, CD40 ligation induces a significant increase in IL-12 p35 and IL-12 p70 heterodimer production. Similarly, IL-12 p70 is produced during T cell activation in the presence but not in the absence of microbial stimuli. Thus, production of bioactive IL-12 by DC can be amplified by T cell-derived signals but must be initiated by innate signals.

The phagocytosis of pathogens is a critical event in host defense, not only for clearance of the invading microorganism, but also for the subsequent immune response. We have examined Dectin-1, a proinflammatory nonopsonic receptor for ␤-glucans, and show that it mediates the internalization of ␤-glucan-bearing ligands, including yeast particles. Although requiring tyrosine phosphorylation and the cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM)-like motif, uptake mediated by Dectin-1 was different from any previously reported phagocytic receptor and was not dependent on Syk-kinase in macrophages. IntroductionPhagocytosis plays a critical role in innate immunity, both by facilitating the removal and killing of pathogens and by priming the adaptive immune response. The phagocytic process is initiated by the cross-linking of an array of dedicated surface receptors, some capable of direct recognition, the so-called pattern recognition receptors (PRRs), and others that recognize opsonins coating the pathogens. Of these receptors, the opsonic Fc␥ (Fc␥Rs) and complement receptors (CRs) are the best described and exhibit different phagocytic mechanisms and subsequent cellular responses that reflect important differences in their signaling pathways. 1,2 Nonopsonic PRRs, such as the macrophage mannose receptor, scavenger receptors, and recently CEACAM3, [3][4][5] have also been suggested to possess phagocytic capacity, but the mechanisms underlying these activities are less clear.We have identified Dectin-1 as the major macrophage PRR for ␤-glucans, carbohydrate polymers that possess anti-infective and antitumorigenic properties in vivo. [6][7][8] Dectin-1, which is also expressed on the surface of other innate immune cells, including neutrophils and dendritic cells, 9 is a C-type lectinlike transmembrane receptor containing an immunoreceptor tyrosine-based activation motif (ITAM)-like motif in its cytoplasmic tail, which becomes tyrosine phosphorylated on ligand binding. 10,11 We and others have shown this motif was required for proinflammatory cytokine production, in collaboration with the Toll-like receptors (TLRs), and for induction of the respiratory burst in response to ␤-glucan ligands, including fungal pathogens. 11,12 The proinflammatory properties of Dectin-1 are similar to those of the ITAM-containing Fc␥Rs, which also mediate the internalization of immune complexes. 13 Phagocytosis by Fc␥R, after ligand binding, is thought to be initiated by src kinase-mediated tyrosine phosphorylation of the receptor ITAM domains leading to the recruitment of p72Syk, a protein tyrosine kinase that is required for subsequent cellular activation and ligand internalization. 1,14 Although the exact downstream pathways leading from Fc␥ and other receptors to actin polymerization and phagocytosis is currently unclear, other molecules, including phosphatidylinositol (PI)-3 kinase, protein kinase C (PKC), and the Rho guanosine triphosphatases (GTPases), are known to be involved. 1 We determined if Dectin-1 could als...

Dendritic cells (DC) can produce Th-polarizing cytokines and direct the class of the adaptive immune response. Microbial stimuli, cytokines, chemokines, and T cell-derived signals all have been shown to trigger cytokine synthesis by DC, but it remains unclear whether these signals are functionally equivalent and whether they determine the nature of the cytokine produced or simply initiate a preprogrammed pattern of cytokine production, which may be DC subtype specific. Here, we demonstrate that microbial and T cell-derived stimuli can synergize to induce production of high levels of IL-12 p70 or IL-10 by individual murine DC subsets but that the choice of cytokine is dictated by the microbial pattern recognition receptor engaged. We show that bacterial components such as CpG-containing DNA or extracts from Mycobacterium tuberculosis predispose CD8α+ and CD8α−CD4− DC to make IL-12 p70. In contrast, exposure of CD8α+, CD4+ and CD8α−CD4− DC to heat-killed yeasts leads to production of IL-10. In both cases, secretion of high levels of cytokine requires a second signal from T cells, which can be replaced by CD40 ligand. Consistent with their differential effects on cytokine production, extracts from M. tuberculosis promote IL-12 production primarily via Toll-like receptor 2 and an MyD88-dependent pathway, whereas heat-killed yeasts activate DC via a Toll-like receptor 2-, MyD88-, and Toll/IL-1R domain containing protein-independent pathway. These results show that T cell feedback amplifies innate signals for cytokine production by DC and suggest that pattern recognition rather than ontogeny determines the production of cytokines by individual DC subsets.

The functional relationships and properties of different subtypes of dendritic cells (DC) remain largely undefined. To better characterize these cells, we used global gene analysis to determine gene expression patterns among murine CD11chigh DC subsets. CD4+, CD8α+, and CD8α− CD4− (double negative (DN)) DC were purified from spleens of normal C57/BL6 mice and analyzed using Affymetrix microarrays. The CD4+ and CD8α+ DC subsets showed distinct basal expression profiles differing by >200 individual genes. These included known DC subset markers as well as previously unrecognized, differentially expressed CD Ags such as CD1d, CD5, CD22, and CD72. Flow cytometric analysis confirmed differential expression in nine of nine cases, thereby validating the microarray analysis. Interestingly, the microarray expression profiles for DN cells strongly resembled those of CD4+ DC, differing from them by <25 genes. This suggests that CD4+ and DN DC are closely related phylogenetically, whereas CD8α+ DC represent a more distant lineage, supporting the historical distinction between CD8α+ and CD8α− DC. However, staining patterns revealed that in contrast to CD4+ DC, the DN subset is heterogeneous and comprises at least two subpopulations. Gene Ontology and literature mining analyses of genes expressed differentially among DC subsets indicated strong associations with immune response parameters as well as cell differentiation and signaling. Such associations offer clues to possible unique functions of the CD11chigh DC subsets that to date have been difficult to define as rigid distinctions.

We present a new, power-free and flexible detection system named MCFphone for portable colorimetric and fluorescence quantitative sandwich immunoassay detection of prostate specific antigen (PSA). The MCFphone is composed by a smartphone integrated with a magnifying lens, a simple light source and a miniaturised immunoassay platform, the Microcapillary Film (MCF). The excellent transparency and flat geometry of fluoropolymer MCF allowed quantitation of PSA in the range 0.9 to 60 ng/ml with<7% precision in 13 min using enzymatic amplification and a chromogenic substrate. The lower limit of detection was further improved from 0.4 to 0.08 ng/ml in whole blood samples with the use of a fluorescence substrate. The MCFphone has shown capable of performing rapid (13 to 22 min total assay time) colorimetric quantitative and highly sensitive fluorescence tests with good %Recovery, which represents a major step in the integration of a new generation of inexpensive and portable microfluidic devices with commercial immunoassay reagents and off-the-shelf smartphone technology.

Dendritic cells (DC) initiate T cell responses and direct the class of T cell immunity through the production of Th‐polarizing cytokines. In the mouse, immunization with CD8α+ DC has led to Th1 priming whereas immunization with CD8α– DC has been associated with Th2 induction. Here, we use a direct T cell priming assay in vitro to re‐examine the Th‐directing potential of total DC or purified CD4+ DC, CD8α+ DC or CD4– CD8α– (double‐negative; DN) DC subsets from mouse spleen. We show that the default Th effector phenotype induced by priming with DC depends on the protocol used for T cell purification, the T cell:antigen‐presenting cell ratio and the antigen dose but is only marginally affected by DC subtype. All DC subsets can direct increased Th1 development in response to microbial stimuli known to elicit IL‐12 production. Similarly, all subsets can suppress Th1 development and allow Th2 cellsto expand upon exposure to IL‐10‐inducing microbial agents. The flexibility of DC in directing Th development in function of microbial signals argues against the notion of pre‐determined "DC1" and "DC2" subsets and suggests that multiple DC subtypes can direct an appropriate Th response to different classes of infectious agents.