Dectin-1 Knockout Research Articles

Dectin1 contributes to hypertensive vascular injury by promoting macrophage infiltration through activating the Syk/NF-κB pathway

Vascular injury is an early manifestation leading to end-organ damage in hypertension pathogenesis, which involves a macrophage-associated immune response. Dendritic cell-associated C-type lectin-1 (Dectin1) is a pivotal player in regulating inflammation-mediated cardiovascular disease. However, its role in hypertension-induced vascular damage and the underlying mechanisms remain unclear. We hypothesized that Dectin1 might accelerate angiotensin II (Ang II)- or deoxycorticosterone acetate–salt (DOCA-salt)-induced vascular injury through proinflammatory actions in macrophages. Macrophage Dectin1 was upregulated in mouse aortic tissues stimulated with Ang II. In the peripheral blood, Ang II also increased CD11b+F4/80+ macrophages in mice. In our constructed Dectin1 knockout mice, Dectin1 deletion protected against Ang II-induced EB extravasation and aortic wall thickness. Deficiency of Dectin1 or its pharmacological inhibition considerably improved fibrosis and inflammation responses, accompanied by a reduction in M1 macrophage polarization as well as proinflammatory cytokines and chemokines induced by Ang II or DOCA-salt. Through the bone marrow (BM) transplantation assay, these effects were verified in the wild type mice reconstituted with Dectin1-deficient BM cells. Mechanistically, Ang II promoted Dectin1 homodimerization, thereby triggering the spleen tyrosine kinase/nuclear factor kappa B pro-inflammatory cascade to induce the expression of inflammatory factors and chemokines in vivo and in vitro. In conclusion, Dectin1 has an essential role in the pathogenic procedure of Ang II-stimulated or DOCA-salt-induced vascular damage in mice and represents a promising therapeutic target for cardiovascular diseases.

252-LB: Dectin-2 Deficiency Promotes Proinflammatory Cytokine Release from Macrophages and Impairs Glucose-Stimulated Insulin Secretion

Pancreatic islet inflammation plays an important role in the pathogenesis of diabetes mellitus. Islet macrophages, which have emerged as a key player in islet inflammation, express Dectin-2 (D2), one of the C-type lectin receptors recognizing α-mannans, while endocrine cells such as α-cells and β-cells lack the expression of D2. To elucidate the role of D2 in glucose metabolism and β-cell function, we analyzed the phenotype of D2 knockout (D2KO) mice. D2KO mice exhibited significantly impaired glucose tolerance compared to wild-type (WT) mice, although there were no significant differences in insulin sensitivity. Glucose-stimulated insulin secretion (GSIS) was significantly reduced in isolated islets from D2KO mice compared to WT mice. Comprehensive analysis of RNA-seq in isolated islets from D2KO and WT mice revealed that D2 deficiency induced immune cell infiltration and inflammation in pancreatic islets. Furthermore, RNA-seq analysis in peritoneal macrophages from D2KO and WT mice showed that macrophages from D2KO mice acquired an inflammatory phenotype and exhibited enhanced NF-κB signaling compared to those from WT mice. Next, to determine whether humoral factors secreted from macrophages affect GSIS, we examined GSIS using the culture supernatants of macrophages from D2KO and WT mice. The culture supernatants of macrophages from D2KO mice suppressed GSIS compared to WT mice. Furthermore, the multiplex cytokine assay revealed that IL-1α and IL-6 were significantly increased in the culture supernatants from macrophages of D2KO mice. Finally, we found that the stimulation of IL-1α and IL-6 reduced GSIS in β-cells. These findings suggest that macrophages from D2KO mice suppress GSIS by increasing IL-1α and IL-6 production. This study provides new insights into the mechanism by which D2 is involved in the pathogenesis of pancreatic islet inflammation. Disclosure M. Fujita: None. T. Miyazawa: None. K. Uchida: None. N. Uchida: None. T. Hatayama: None. S. Nakamura: None. Y. Takeichi: None. Y. Miyachi: None. Y. Ogawa: None.

Dectin-1 Acts as a Non-Classical Receptor of Ang II to Induce Cardiac Remodeling.

Cardiac remodeling in heart failure involves macrophage-mediated immune responses. Recent studies have shown that a PRR (pattern recognition receptor) called dectin-1, expressed on macrophages, mediates proinflammatory responses. Whether dectin-1 plays a role in pathological cardiac remodeling is unknown. Here, we identified a potential role of dectin-1 in this disease. To model aberrant cardiac remodeling, we utilized mouse models of chronic Ang II (angiotensin II) infusion. In this model, we assessed the potential role of dectin-1 through using D1KO (dectin-1 knockout) mice and bone marrow transplantation chimeric mice. We then used cellular and molecular assays to discover the underlying mechanisms of dectin-1 function. We found that macrophage dectin-1 is elevated in mouse heart tissues following chronic Ang II administration. D1KO mice were significantly protected against Ang II-induced cardiac dysfunction, hypertrophy, fibrosis, inflammatory responses, and macrophage infiltration. Further bone marrow transplantation studies showed that dectin-1 deficiency in bone marrow-derived cells prevented Ang II-induced cardiac inflammation and dysfunction. Through detailed molecular studies, we show that Ang II binds directly to dectin-1, causing dectin-1 homodimerization and activating the downstream Syk (spleen tyrosine kinase)/NF-κB (nuclear factor kappa B) signaling pathway to induce expression of inflammatory and chemoattractant factors. Mutagenesis studies identified R184 in the C-type lectin domain to interact with Ang II. Blocking dectin-1 in macrophages suppresses Ang II-induced inflammatory mediators and subsequent intercellular cross talk with cardiomyocytes and fibroblasts. Our study has discovered dectin-1 as a new nonclassical receptor of Ang II and a key player in cardiac remolding and dysfunction. These studies suggest that dectin-1 may be a new target for treating hypertension-related heart failure.

Deletion of both Dectin-1 and Dectin-2 affects the bacterial but not fungal gut microbiota and susceptibility to colitis in mice

BackgroundInnate immunity genes have been reported to affect susceptibility to inflammatory bowel diseases (IBDs) and colitis in mice. Dectin-1, a receptor for fungal cell wall β-glucans, has been clearly implicated in gut microbiota modulation and modification of the susceptibility to gut inflammation. Here, we explored the role of Dectin-1 and Dectin-2 (another receptor for fungal cell wall molecules) deficiency in intestinal inflammation.DesignSusceptibility to dextran sodium sulfate (DSS)-induced colitis was assessed in wild-type, Dectin-1 knockout (KO), Dectin-2KO, and double Dectin-1KO and Dectin-2KO (D-1/2KO) mice. Inflammation severity, as well as bacterial and fungal microbiota compositions, was monitored.ResultsWhile deletion of Dectin-1 or Dectin-2 did not have a strong effect on DSS-induced colitis, double deletion of Dectin-1 and Dectin-2 significantly protected the mice from colitis. The protection was largely mediated by the gut microbiota, as demonstrated by fecal transfer experiments. Treatment of D-1/2KO mice with opportunistic fungal pathogens or antifungal agents did not affect the protection against gut inflammation, suggesting that the fungal microbiota had no role in the protective phenotype. Amplicon-based microbiota analysis of the fecal bacterial and fungal microbiota of D-1/2KO mice confirmed the absence of changes in the mycobiota but strong modification of the bacterial microbiota. We showed that bacteria from the Lachnospiraceae family were at least partly involved in this protection and that treatment with Blautia hansenii was enough to recapitulate the protection.ConclusionsDeletion of both the Dectin-1 and Dectin-2 receptors triggered a global shift in the microbial gut environment, affecting, surprisingly, mainly the bacterial population and driving protective effects in colitis. Members of the Lachnospiraceae family seem to play a central role in this protection. These findings provide new insights into the role of the Dectin receptors, which have been described to date as affecting only the fungal population, in intestinal physiopathology and in IBD.F446R2nzvxqjjpdjAkXVZkVideo

Insights on the Functional Role of Beta-Glucans in Fungal Immunity Using Receptor-Deficient Mouse Models.

Understanding the host anti-fungal immunity induced by beta-glucan has been one of the most challenging conundrums in the field of biomedical research. During the last couple of decades, insights on the role of beta-glucan in fungal disease progression, susceptibility, and resistance have been greatly augmented through the utility of various beta-glucan cognate receptor-deficient mouse models. Analysis of dectin-1 knockout mice has clarified the downstream signaling pathways and adaptive effector responses triggered by beta-glucan in anti-fungal immunity. On the other hand, assessment of CR3-deficient mice has elucidated the compelling action of beta-glucans in neutrophil-mediated fungal clearance, and the investigation of EphA2-deficient mice has highlighted its novel involvement in host sensing and defense to oral mucosal fungal infection. Based on these accounts, this review focuses on the recent discoveries made by these gene-targeted mice in beta-glucan research with particular emphasis on the multifaceted aspects of fungal immunity.

Dectin-2-mediated initiation of immune responses caused by influenza virus hemagglutinin.

Antigen-presenting cells express pattern recognition receptors (PRRs), which sense pathogen-associated molecular patterns from microorganisms and lead to the induction of inflammatory responses. C-type lectin receptors (CLRs), the representative PRRs, bind to microbial polysaccharides, among which Dectin-2 and Mincle recognize mannose-containing polysaccharides. Because influenza virus (IFV) hemagglutinin (HA) is rich in mannose polysaccharides, Dectin-2 or Mincle may contribute to the recognition of HA. In this study, we addressed the possible involvement of Dectin-2 and Mincle in the viral recognition and the initiation of cytokine production. Interleukin (IL)-12p40 and IL-6 production by bone marrow-derived dendritic cells (BM-DCs) upon stimulation with HA was significantly reduced in Dectin-2 knockout (KO) mice compared to wild-type (WT) mice whereas there was no difference between WT mice and Mincle KO mice. BM-DCs that were treated with Syk inhibitor resulted in a significant reduction of cytokine production upon stimulation with HA. The treatment of BM-DCs with methyl-α-D-mannopyranoside (ManP) also led to a significant reduction in cytokine production by BM-DCs that were stimulated with HA, except for the A/H1N1pdm09 subtype. IL-12p40 and IL-6 synthesis by BM-DCs was completely diminished upon stimulation with HA treated with concanavalin A (ConA)-bound sepharose beads. Finally, GFP expression was detected in reporter cells that were transfected with the Dectin-2 gene, but not with the Mincle gene, when stimulated with HA derived from the A/H3N2 subtype. These data suggested that Dectin-2 may be a key molecule as the sensor for IFV to initiate the immune response and regulate the pathogenesis of IFV infection.

Effect of CARD9 Deficiency on Neutrophil-Mediated Host Defense against Pulmonary Infection with Streptococcus pneumoniae.

Streptococcus pneumoniae is a major causative bacterium of community-acquired pneumonia. Dendritic cell-associated C-type lectin-2 (dectin-2), one of the C-type lectin receptors (CLRs), was previously reported to play a pivotal role in host defense against pneumococcal infection through regulating phagocytosis by neutrophils while not being involved in neutrophil accumulation. In the present study, to elucidate the possible contribution of other CLRs to neutrophil accumulation, we examined the role of caspase recruitment domain-containing protein 9 (CARD9), a common adaptor molecule for signal transduction triggered by CLRs, in neutrophilic inflammatory response against pneumococcal infection. Wild-type (WT), CARD9 knockout (KO), and dectin-2 KO mice were infected intratracheally with pneumococcus, and the infected lungs were histopathologically analyzed to assess neutrophil accumulation at 24 h postinfection. Bronchoalveolar lavage fluids (BALFs) were collected at the same time point to count the neutrophils and assess the production of inflammatory cytokines and chemokines. Neutrophil accumulation was significantly decreased in CARD9 KO mice, but not in dectin-2 KO mice. Tumor necrosis factor alpha (TNF-α), keratinocyte-derived chemokine (KC), and macrophage inflammatory protein-2 (MIP-2) production in BALFs were also attenuated in CARD9 KO mice, but not in dectin-2 KO mice. Production of TNF-α and KC by alveolar macrophages stimulated with pneumococcal culture supernatants was significantly attenuated in CARD9 KO mice, but not in dectin-2 KO mice, compared to that in each group's respective control mice. In addition, pneumococcus-infected CARD9 KO mice showed larger bacterial burdens in the lungs than did WT mice. These data indicate that CARD9 is required for neutrophil migration after pneumococcal infection, as well as inflammatory cytokine and chemokine production by alveolar macrophages, and suggest that a CLR distinct from dectin-2 may be involved in this response.

TLR4, but Neither Dectin-1 nor Dectin-2, Participates in the Mollusk Hemocyanin-Induced Proinflammatory Effects in Antigen-Presenting Cells From Mammals.

Mollusk hemocyanins have biomedical uses as carriers/adjuvants and nonspecific immunostimulants with beneficial clinical outcomes by triggering the production of proinflammatory cytokines in antigen-presenting cells (APCs) and driving immune responses toward type 1 T helper (Th1) polarization. Significant structural features of hemocyanins as a model antigen are their glycosylation patterns. Indeed, hemocyanins have a multivalent nature as highly mannosylated antigens. We have previously shown that hemocyanins are internalized by APCs through receptor-mediated endocytosis with proteins that contain C-type lectin domains, such as mannose receptor (MR). However, the contribution of other innate immune receptors to the proinflammatory signaling pathway triggered by hemocyanins is unknown. Thus, we studied the roles of Dectin-1, Dectin-2, and Toll-like receptor 4 (TLR4) in the hemocyanin activation of murine APCs, both in dendritic cells (DCs) and macrophages, using hemocyanins from Megathura crenulata (KLH), Concholepas concholepas (CCH) and Fissurella latimarginata (FLH). The results showed that these hemocyanins bound to chimeric Dectin-1 and Dectin-2 receptors in vitro; which significantly decreased when the glycoproteins were deglycosylated. However, hemocyanin-induced proinflammatory effects in APCs from Dectin-1 knock-out (KO) and Dectin-2 KO mice were independent of both receptors. Moreover, when wild-type APCs were cultured in the presence of hemocyanins, phosphorylation of Syk kinase was not detected. We further showed that KLH and FLH induced ERK1/2 phosphorylation, a key event involved in the TLR signaling pathway. We confirmed a glycan-dependent binding of hemocyanins to chimeric TLR4 in vitro. Moreover, DCs from mice deficient for MyD88-adapter-like (Mal), a downstream adapter molecule of TLR4, were partially activated by FLH, suggesting a role of the TLR pathway in hemocyanin recognition to activate APCs. The participation of TLR4 was confirmed through a decrease in IL-12p40 and IL-6 secretion induced by FLH when a TLR4 blocking antibody was used; a reduction was also observed in DCs from C3H/HeJ mice, a mouse strain with a nonfunctional mutation for this receptor. Moreover, IL-6 secretion induced by FLH was abolished in macrophages deficient for TLR4. Our data showed the involvement of TLR4 in the hemocyanin-mediated proinflammatory response in APCs, which could cooperate with MR in innate immune recognition of these glycoproteins.

Differential susceptibility of Dectin-1 isoforms to functional inactivation by neutrophil and fungal proteases.

Patients with cystic fibrosis (CF) experience chronic or recurrent bacterial and fungal lung infections. Many patients with CF cannot effectively clear Aspergillus from their lungs. This may result in IgE sensitization and the development of allergic bronchopulmonary aspergillosis, or invasive infections, such as Aspergillus bronchitis. Lung disease in patients with CF is associated with neutrophil-dominated inflammation and elevated levels of the serine protease, neutrophil elastase (NE). Various C-type lectin-like receptors (CLRs), including Dectin-1 and Dectin-2, are involved in the immune response to Aspergillus. Here, we show that purified NE cleaves Dectin-1 in an isoform-specific manner. Bronchoalveolar lavage fluid from patients with CF, which contains high NE activity, induces Dectin-1 cleavage. Similarly, filtrate from a protease-producing strain of Aspergillus fumigatus induces isoform-specific cleavage of Dectin-1. Dectin-1 knockout (KO) cells and NE-treated cells demonstrated reduced phagocytosis of zymosan, a fungal cell wall preparation. In addition, NE cleaves 2 other CLRs, Dectin-2 and Mincle, and fungal-induced cytokine production was reduced in Dectin-1 KO cells, Dectin-2 KO cells, and NE-treated cells. Thus, Dectin-1 and Dectin-2 cleavage by NE and/or A. fumigatus–derived proteases results in an aberrant antifungal immune response that likely contributes to disease pathology in patients with CF.—Griffiths, J. S., Thompson, A., Stott, M., Benny, A., Lewis, N. A., Taylor, P. R., Forton, J., Herrick, S., Orr, S. J., McGreal, E. P. Differential susceptibility of Dectin-1 isoforms to functional inactivation by neutrophil and fungal proteases.

Lentinan Exerts its Anti-Inflammatory Activity by Suppressing TNFR1 Transfer to the Surface of Intestinal Epithelial Cells through Dectin-1 in an in vitro and mice model

It has been reported that lentinan, β-1,3; 1,6-glucan derived from Lentinula edodes, suppresses intestinal inflammation and ameliorates symptoms of colitis. However, the mechanism of intestinal anti-inflammatory activity of lentinan and how it is recognized by intestinal epithelial cells remains largely unclear. The receptor for lentinan on intestinal epithelial cells was identified using an in vitro gut inflammation model consisting of Caco-2 and RAW264.7 cells. Colitis was induced in 7 to 8 week-old wild-type (WT) or knockout (KO) mice by the free intake of water containing 2% dextran sulfate sodium (DSS) for 7 days. Lentinan was administered daily via intragastric administration. Tumor necrosis factor receptor 1 (TNFR1)-GFP complex was constructed to monitor its movement in Caco-2 cells using confocal and total internal fluorescence microscopy. The results indicated that lentinan suppressed DSS-induced body weight loss, shortening of colon length, histological score, and inflammatory cytokine mRNA expression in the inflamed tissues of WT mice, whereas these suppressive effects of lentinan were not observed in Dectin-1 KO mice. Furthermore, lentinan reduced TNFR1 expression in intestinal epithelial cells of WT mice but not those from Dectin-1 KO mice. Using TNFR1-GFP constructs, it was confirmed that lentinan reduced TNFR1 expression on Caco-2 cell membranes through Dectin-1 ligation. Our study revealed that lentinan suppressed intestinal inflammation by Dectin-1-mediated regulation of TNFR1 transfer to the surface of intestinal epithelial cells.

Zymosan-Induced Peritonitis: Effects on Cardiac Function, Temperature Regulation, Translocation of Bacteria, and Role of Dectin-1.

Zymosan-induced peritonitis is a model commonly used to study systemic inflammatory response syndrome and multiple organ dysfunction syndrome. However, effects of zymosan on cardiac function have not been reported. We evaluated cardiac responses to zymosan in mice and the role of β-glucan and dectin-1 in mediating these responses. Temperature and cardiac function were evaluated before and after intraperitoneal (i.p.) injection of zymosan (100 or 500 mg/kg) or saline. Chronotropic and dromotropic functions were measured using electrocardiograms (ECGs) collected from conscious mice. Cardiac inotropic function was determined by echocardiography. High-dose zymosan caused a rapid and maintained hypothermia along with visual signs of illness. Baseline heart rate (HR) was unaffected but HR variability (HRV) increased, and there was a modest slowing of ventricular conduction. High-dose zymosan also caused prominent decreases in cardiac contractility at 4 and 24 h. Because zymosan is known to cause gastrointestinal tract pathology, peritoneal wash and blood samples were evaluated for bacteria at 24 h after zymosan or saline injection. Translocation of bacterial occurred in all zymosan-treated mice (n = 3), and two had bacteremia. Purified β-glucan (50 and 125 mg/kg, i.p.) had no effect on temperature or ECG parameters. However, deletion of dectin-1 modified the ECG responses to high-dose zymosan; slowing of ventricular conduction and the increase in HRV were eliminated but a marked bradycardia appeared at 24 h after zymosan treatment. Zymosan-treated dectin-1 knockout mice also showed hypothermia and visual signs of illness. Fecal samples from dectin-1 knockout mice contained more bacteria than wild types, but zymosan caused less translocation of bacteria. Collectively, these findings demonstrate that zymosan-induced systemic inflammation causes cardiac dysfunction in mice. The data suggest that dectin-1-dependent and -independent mechanisms are involved. Although zymosan treatment causes translocation of bacteria, this effect does not have a major role in the overall systemic response to zymosan.

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.

Neither Dectin-2 nor the Mannose Receptor Is Required for Resistance to Coccidioides immitis in Mice

We investigated the roles of the mannose receptor (MR) and Dectin-2 in resistance to pulmonary coccidioidomycosis in C57BL/6 (B6) mice and in the interaction of myeloid cells with spherules, using B6 mice with targeted mutations in Mrc1 and Clec4n. Spherules are the tissue form of Coccidioides, and we determined that the MR on bone marrow-derived dendritic cells (BMDC) was important for recognition of spherules (formalin-killed spherules [FKS]) and for secretion of interleukin 10 (IL-10) and proinflammatory cytokines in response to FKS by both elicited macrophages and BMDC. Infected MR knockout (KO) mice produced more IL-10 in their lungs than did B6 mice, and MR KO mice also made more protective Th-17 cytokines. In contrast to the MR, Dectin-2 was not required for recognition of FKS by BMDC or for the production of cytokines by BMDC in response to FKS. However, Dectin-2 KO was required for stimulation of elicited peritoneal macrophages. Despite that, lung cytokine levels were not significantly different in Dectin-2 KO mice and B6 mice 14 days after infection, except for IL-1β, which was higher in Dectin-2 KO lungs. Although both Dectin-2(-/-) and MR(-/-) myeloid cells had reduced proinflammatory cytokine responses to FKS in vitro, neither MR nor Dectin-2 deficiency reduced the resistance of B6 mice to pulmonary coccidioidomycosis.

Dectin-2-Dependent NKT Cell Activation and Serotype-Specific Antibody Production in Mice Immunized with Pneumococcal Polysaccharide Vaccine

Although thymus-independent type 2 antigens generally do not undergo Ig class switching from IgM to IgG, pneumococcal polysaccharide vaccine (PPV) induces the production of serotype-specific IgG. How this happens remains unclear, however. In the present study, PPV immunization induced production of IgG as well as IgM specific for a serotype 3-pneumococcal polysaccharide in the sera of wild-type (WT) mice, but this phenomenon was significantly reduced in Dectin-2 knockout (KO) mice. Immunization with PPV caused IL-12p40 production in WT mice, but this response was significantly reduced in Dectin-2KO mice. Likewise, immunization with PPV activated natural killer T (NKT) cells in WT mice but not in Dectin-2KO mice. Furthermore, administration of α-galactosylceramide, recombinant (r)IL-12 or rIFN-γ improved the reduced IgG levels in Dectin-2KO mice, and treatment with neutralizing anti-IFN-γ mAb resulted in the reduction of IgG synthesis in PPV-immunized WT mice. Transfer of spleen cells from PPV-immunized WT mice conferred protection against pneumococcal infection on recipient mice, whereas this effect was cancelled when the transferred spleen cells were harvested from PPV-immunized Dectin-2KO mice. These results suggest that the detection of PPV antigens via Dectin-2 triggers IL-12 production, which induces IFN-γ synthesis by NKT cells and subsequently the production of serotype-specific IgG.

199: Beta-glucan inducible microRNAs negatively regulate inflammatory response

Beta-glucan is key structural component of yeast cell wall. Although the inflammatory response and signaling events following beta-glucan receptor dectin-1 engagement have been relatively well described, the effect of beta-glucans on leukocyte microRNA expression has not been previously characterized in detail. We performed microRNA expression array using primary human macrophages stimulated with beta-glucan Curdlan. After qPCR validation we identified that beta-glucan increased the expression of miR-29b1-5p, miR-132, miR146a, miR-155 and miR-212. In follow-up experiments using various microbial ligands, we observed that miR29b1-5p, miR-132 and miR-212 were specifically induced by beta-glucans while the well characterized inflammatory miR-146a and miR-155 were induced by all stimuli. The sequences of beta-glucan inducible microRNAs are conserved in both human and mouse. To gain in vivo insight into beta-glucan inducible microRNAs we used mouse beta-glucan inhalation model and noticed that the beta-glucan microRNAs were also up-regulated in vivo in mouse lung leukocytes. Moreover, experiments using dectin-1 knockout mice revealed that microRNA expression was dectin-1 dependent. Bioinformatic analysis of microRNA target proteins suggested that beta-glucan inducible microRNAs might regulate inflammatory signaling events. Our functional in vitro experiments revealed that beta-glucan inducible miRNAs act as negative regulators of immune response. More specifically, overexpression of miR-29b1-5p, miR-132 or miR-155 reduced beta-glucan-induced production of IL-1beta and TNF-alpha, while inhibition of these microRNAs during beta-glucan stimulation augmented cytokine response. Although the exact molecular mechanisms are still under investigation, our preliminary findings suggest that modulation of inflammatory response could act via SIRT1, which has been shown to regulate multiple aspects of inflammatory response. In conclusion we show here that beta-glucan induces expression of both unique and common inflammatory microRNAs and that these microRNAs form a negative feedback loop that limits extensive inflammatory response.

Role of Dectin-2 in the serotype-specific antibody production and host protection against Streptococcus pneumoniae infection caused by pneumococcal polysaccharide vaccine (166.8)

Abstract Anti-pneumococcal polysaccharide vaccine (PPV) induces the production of serotype-specific Ab including IgG2 in spite of its feature of thymus-independent antigen. It remains to be clarified how PPV causes a class-switching from IgM to IgG. Recently, C-type lectin receptors (CLR) sensing polysaccharides have garnered much attention in the host defense to fungal infection. In our recent study, Dectin-2, a CLR involved in the recognition of mannose-rich polysaccharides, has been found to play an important role in the host defense to pneumococcal infection. Therefore, in this study, we addressed how Dectin-2 was involved in the vaccine effect of PPV using a mouse model. The serum levels of anti-serotype 3 pneumococcal capsular polysaccharide (PPS-3) two weeks after vaccination were significantly reduced in Dectin-2 knockout (KO) mice compared to wild-type (WT) mice. Weight loss and hypothermia of mice caused by infection with Streptococcus pneumoniae (URF918) were improved in WT mice transferred with spleen cells from vaccinated WT mice compared to those from vaccinated Dectin-2KO mice. Similar tendency was observed in the survival of infected mice. In in vitro experiments, IL-12p40, TNF-α and IL-6 production by bone marrow-derived dendritic cells (BM-DCs) stimulated with PPV was abrogated in Dectin-2KO. These results suggest that Dectin-2 is involved in the PPV-induced serotype-specific Ab production and host protection through sensing the pneumococcal polysaccharide antigens.

Dectin-1 Y238X polymorphism associates with susceptibility to invasive aspergillosis in hematopoietic transplantation through impairment of both recipient- and donor-dependent mechanisms of antifungal immunity

AbstractThe C-type lectin receptor Dectin-1 plays a pivotal role in antifungal immunity. In this study, the recently characterized human DECTIN1 Y238X early stop codon polymorphism leading to diminished Dectin-1 receptor activity was studied in relation to invasive aspergillosis susceptibility and severity in patients receiving hematopoietic stem cell transplantation. We found that the presence of the DECTIN1 Y238X polymorphism in either donors or recipients of hematopoietic stem cell transplantation increased susceptibility to aspergillosis, with the risk being highest when the polymorphism was present simultaneously in both donors and recipients (adjusted hazard ratio = 3.9; P = .005). Functionally, the Y238X polymorphism impaired the production of interferon-γ and interleukin-10 (IL-10), in addition to IL-1β, IL-6, and IL-17A, by human peripheral mononuclear cells and Dectin-1 on human epithelial cells contributed to fungal recognition. Mechanistically, studies on preclinical models of infection in intact or bone marrow-transplanted Dectin-1 knockout mice revealed that protection from infection requires a distinct, yet complementary, role of both donor and recipient Dectin-1. This study discloses Dectin-1 deficiency as a novel susceptibility factor for aspergillosis in high-risk patients and identifies a previously unsuspected role for Dectin-1 in antifungal immunity that is the ability to control both resistance and tolerance to the fungus contingent on hematopoietic/nonhematopoietic compartmentalization.

OR.19. TNFR1 Controls the Recruitment of Macrophages but Not T Cells in 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.

Contribution of dectin-1 and granulocyte macrophage–colony stimulating factor (GM-CSF) to immunomodulating actions of β-glucan

β-Glucans are major cell wall structural components in fungi. As they are not found in animals, these carbohydrates are considered to be classic pathogen-associated molecular patterns (PAMPs), and are recognized by the innate immune system. Although their immunomodulating activities have been shown to be associated with the recognition of some fungi, and with their medicinal properties in the field of cancer immunotherapy, it is still unclear how β-glucans mediate their effects. Recent studies have started to shed some light on their cellular receptors, such as dectin-1, and their molecular mechanisms of action. We have extensively investigated the response of leukocytes to β-glucan, focusing on cytokine induction by SCG, which is a major 6-branched 1,3-β-d-glucan in Sparassis crispa Fr. There is a strain difference in the reactivity of mice to SCG, and DBA/1 and DBA/2 mice are highly sensitive strains. In the process of research on cytokine induction by SCG in DBA/2 mice, we found that GM-CSF plays a key biological role in this activity. Cytokine induction by SCG was completely abolished in dendritic cells from dectin-1 knockout mice. On the other hand, controlling the level of endogenous GM-CSF production and/or dectin-1 expression could regulate the reactivity to β-glucan. These results indicate that the key factors in the responsiveness to β-glucan are GM-CSF production and dectin-1 expression. In this review, we describe how the key molecules related to the expression of the immunomodulating activities of β-glucan were identified, and how the response to β-glucan is controlled.