The ability of fungal-derived β-glucan particles to induce leukocyte activation and the production of inflammatory mediators, such as tumor necrosis factor (TNF)-α, is a well characterized phenomenon. Although efforts have been made to understand how these carbohydrate polymers exert their immunomodulatory effects, the receptors involved in generating these responses are unknown. Here we show that Dectin-1 mediates the production of TNF-α in response to zymosan and live fungal pathogens, an activity that occurs at the cell surface and requires the cytoplasmic tail and immunoreceptor tyrosine activation motif of Dectin-1 as well as Toll-like receptor (TLR)-2 and Myd88. This is the first demonstration that the inflammatory response to pathogens requires recognition by a specific receptor in addition to the TLRs. Furthermore, these studies implicate Dectin-1 in the production of TNF-α in response to fungi, a critical step required for the successful control of these pathogens.

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...

Among patients without substantial lung entrapment, the outpatient administration of talc through an indwelling pleural catheter for the treatment of malignant pleural effusion resulted in a significantly higher chance of pleurodesis at 35 days than an indwelling catheter alone, with no deleterious effects. (Funded by Becton Dickinson; EudraCT number, 2012-000599-40 .).

We recently demonstrated that the β-glucan receptor Dectin-1 (βGR) was the major nonopsonic β-glucan receptor on macrophages (Mφ) for the yeast-derived particle zymosan. However, on resident peritoneal Mφ, we identified an additional mannan-inhibitable receptor for zymosan that was distinct from the Mφ mannose receptor (MR). In this study, we have studied the mannose-binding potential of murine Mφ and identified the dendritic cell-specific ICAM-3-grabbing nonintegrin homolog, SIGN-related 1 (SIGNR1), as a major MR on murine resident peritoneal Mφ. Both SIGNR1 and βGR cooperated in the nonopsonic recognition of zymosan by these Mφ. When SIGNR1 was introduced into NIH3T3 fibroblasts or RAW 264.7 Mφ, it conferred marked zymosan-binding potential on these cells. However, in the nonprofessional phagocytes (NIH3T3), SIGNR1 was found to be poorly phagocytic, suggesting that other receptors such as βGR may play a more dominant role in particle internalization on professional phagocytes. Binding of zymosan to RAW 264.7 Mφ expressing SIGNR1 resulted in TNF-α production. Treatment of RAW 264.7 Mφ expressing SIGNR1, which express low levels of βGR, with β-glucans had little effect on binding or TNF-α production, indicating that there was no absolute requirement for βGR in this process. These studies have identified SIGNR1 as a major MR for fungal and other pathogens present on specific subsets of Mφ.

Rationale: Acute respiratory distress syndrome is refractory to pharmacological intervention. Inappropriate activation of alveolar neutrophils is believed to underpin this disease's complex pathophysiology, yet these cells have been little studied.Objectives: To examine the functional and transcriptional profiles of patient blood and alveolar neutrophils compared with healthy volunteer cells, and to define their sensitivity to phosphoinositide 3-kinase inhibition.Methods: Twenty-three ventilated patients underwent bronchoalveolar lavage. Alveolar and blood neutrophil apoptosis, phagocytosis, and adhesion molecules were quantified by flow cytometry, and oxidase responses were quantified by chemiluminescence. Cytokine and transcriptional profiling were used in multiplex and GeneChip arrays.Measurements and Main Results: Patient blood and alveolar neutrophils were distinct from healthy circulating cells, with increased CD11b and reduced CD62L expression, delayed constitutive apoptosis, and primed oxidase responses. Incubating control cells with disease bronchoalveolar lavage recapitulated the aberrant functional phenotype, and this could be reversed by phosphoinositide 3-kinase inhibitors. In contrast, the prosurvival phenotype of patient cells was resistant to phosphoinositide 3-kinase inhibition. RNA transcriptomic analysis revealed modified immune, cytoskeletal, and cell death pathways in patient cells, aligning closely to sepsis and burns datasets but not to phosphoinositide 3-kinase signatures.Conclusions: Acute respiratory distress syndrome blood and alveolar neutrophils display a distinct primed prosurvival profile and transcriptional signature. The enhanced respiratory burst was phosphoinositide 3-kinase-dependent but delayed apoptosis and the altered transcriptional profile were not. These unexpected findings cast doubt over the utility of phosphoinositide 3-kinase inhibition in acute respiratory distress syndrome and highlight the importance of evaluating novel therapeutic strategies in patient-derived cells.

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A complete understanding of phagocytosis requires insight into both its biochemical and physical aspects. One of the ways to explore the physical mechanism of phagocytosis is to probe whether and how the target properties (e.g., size, shape, surface states, stiffness, etc.) affect their uptake. Here we report an imaging-based method to explore phagocytosis kinetics, which is compatible with real-time imaging and can be used to validate existing reports using fixed and stained cells. We measure single-event engulfment time from a large number of phagocytosis events to compare how size and shape of targets determine their engulfment. The data shows an increase in the average engulfment time for increased target size, for spherical particles. The uptake time data on nonspherical particles confirms that target shape plays a more dominant role than target size for phagocytosis: Ellipsoids with an eccentricity of 0.954 and much smaller surface areas than spheres were taken up five times more slowly than spherical targets.

Summary Cells of the innate immune system express a large repertoire of germ‐line encoded cell‐surface glycoprotein receptors including Toll‐like receptors (TLRs). TLRs recognize conserved motifs on microbes and induce inflammatory signals. Evidence suggests that individual members of the TLR family or other non‐TLR surface antigens either physically or functionally interact with each other and cumulative effects of these interactions instruct the nature and outcome of the immune response to a particular pathogen.