MBL-mediated opsonophagocytosis of Candida albicans by human neutrophils is coupled with intracellular Dectin-1-triggered ROS production.

Mannan-binding lectin (MBL), a lectin homologous to C1q, greatly facilitates C3/C4-mediated opsonophagocytosis of Candida albicans (C. albicans) by human neutrophils, and has the capacity to bind to CR1 (CD35) expressed on circulating neutrophils. The intracellular pool of neutrophil Dectin-1 plays a critical role in stimulating the reactive oxygen species (ROS) generation through recognition of β-1,3-glucan component of phagocytized zymosan or yeasts. However, little is known about whether MBL can mediate the opsonophagocytosis of Candida albicans by neutrophils independent of complement activation, and whether MBL-mediated opsonophagocytosis influence the intracellular expression of Dectin-1 and ROS production. Here we showed that the inhibited phagocytic efficiency of neutrophils as a result of blockage of Dectin-1 was compensated by exogenous MBL alone in a dose-dependent manner. Furthermore, the expressions of Dectin-1 at mRNA and intracellular protein levels were significantly up-regulated in neutrophils stimulated by MBL-pre-incubated C. albicans, while the expression of surface Dectin-1 remained almost unchanged. Nevertheless, the stimulated ROS production in neutrophils was partly and irreversibly inhibited by blockage of Dectin-1 in the presence of exogenous MBL. Confocal microscopy examination showed that intracellular Dectin-1 was recruited and co-distributed with ROS on the surface of some phagocytized yeasts. The β-1,3-glucanase digestion test further suggested that the specific recognition and binding site of human Dectin-1 is just the β-1,3-glucan moiety on the cell wall of C. albicans. These data demonstrate that MBL has an ability to mediate the opsonophagocytosis of Candida albicans by human neutrophils independent of complement activation, which is coupled with intracellular Dectin-1-triggered ROS production

Transformation of Fonsecaea pedrosoi into sclerotic cells links to the refractoriness of experimental chromoblastomycosis in BALB/c mice via a mechanism involving a chitin-induced impairment of IFN-gamma production

Fonsecaea pedrosoi (F. pedrosoi) is the most common agent of chromoblastomycosis. Transformation of this fungus from its saprophytic phase into pathogenic sclerotic cells in tissue is an essential link to the refractoriness of this infection. Experimental studies in murine models have shown that the absence of CD4+ T cells impairs host defense against F. pedrosoi infection. Clinical research has also suggested that a relatively low level of the Th1 cytokine INF-gamma and inefficient T cell proliferation are simultaneously present in patients with severe chromoblastomycosis upon in vitro stimulation with ChromoAg, an antigen prepared from F. pedrosoi. In the present study, we show that in mice intraperitoneally infected with F. pedrosoi-spores, -hyphae or in vitro-induced sclerotic cells respectively, the transformation of this causative agent into sclerotic cells contributes to a compromised Th1 cytokine production in the earlier stage of infection with impaired generation of neutrophil reactive oxygen species (ROS) and pan-inhibition of Th1/Th2/Th17 cytokine production with disseminated infection in the later stage by using a CBA murine Th1/Th2/Th17 cytokine kit. In addition, we have further demonstrated that intraperitoneal administration of recombinant mouse IFN-gamma (rmIFN-gamma) effectively reduces the fungal load in the infected mouse spleen, and dampens the peritoneal dissemination of F. pedrosoi-sclerotic cells. Meanwhile, exogeneous rmIFN-gamma contributes to the formation and maintenance of micro-abscess and restores the decrease in neutrophil ROS generation in the mouse spleen infected with F. pedrosoisclerotic cells. Of note, we have once again demonstrated that it is a chitin-like component, but not beta-glucans or mannose moiety, that exclusively accumulates on the outer cell wall of F. pedrosoi-sclerotic cells which were induced in vitro or isolated from the spleens of intraperitoneally infected BALB/c mice. In addition, our results indicate that decreased accumulation of chitin on the surface of live F. pedrosoi-sclerotic cells after chitinase treatment can be self-compensated in a time-dependent manner. Importantly, we have for the first time demonstrated that exclusive accumulation of chitin on the transformed sclerotic cells of F. pedrosoi is involved in an impaired murine Th1 cytokine profile, therefore promoting the refractoriness of experimental murine chromoblastomycosis

A chitin-like component on sclerotic cells of Fonsecaea pedrosoi inhibits Dectin-1-mediated murine Th17 development by masking β-glucans.

Fonsecaea pedrosoi (F. pedrosoi), a major agent of chromoblastomycosis, has been shown to be recognized primarily by C-type lectin receptors (CLRs) in a murine model of chromoblastomycosis. Specifically, the β-glucan receptor, Dectin-1, mediates Th17 development and consequent recruitment of neutrophils, and is evidenced to have the capacity to bind to saprophytic hyphae of F. pedrosoi in vitro. However, when embedded in tissue, most etiological agents of chromoblastomycosis including F. pedrosoi will transform into the sclerotic cells, which are linked to the greatest survival of melanized fungi in tissue. In this study, using immunocompetent and athymic (nu/nu) murine models infected subcutaneously or intraperitoneally with F. pedrosoi, we demonstrated that T lymphocytes play an active role in the resolution of localized footpad infection, and there existed a significantly decreased expression of Th17-defining transcription factor Rorγt and inefficient recruitment of neutrophils in chronically infected spleen where the inoculated mycelium of F. pedrosoi transformed into the sclerotic cells. We also found that Dectin-1-expressing histocytes and neutrophils participated in the enclosure of transformed sclerotic cells in the infectious foci. Furthermore, we induced the formation of sclerotic cells in vitro, and evidenced a significantly decreased binding capacity of human or murine-derived Dectin-1 to the induced sclerotic cells in comparison with the saprophytic mycelial forms. Our analysis of β-glucans-masking components revealed that it is a chitin-like component, but not the mannose moiety on the sclerotic cells, that interferes with the binding of β-glucans by human or murine Dectin-1. Notably, we demonstrated that although Dectin-1 contributed to the development of IL-17A-producing CD3+CD4+ murine splenocytes upon in vitro-stimulation by saprophytic F. pedrosoi, the masking effect of chitin components partly inhibited Dectin-1-mediated Th17 development upon in vitro-stimulation by induced sclerotic cells. Therefore, these findings extend our understanding of the chronicity of chromoblastomycosis

Intracellular expression of Dectin-1 was co-distributed with stimulated ROS on the <i>C. albicans</i> which was pre-incubated with MBL and phagocytized by human neutrophils.

<p><b>A </b><b>and B</b>. Localization of Dectin-1 expression and stimulated ROS were determined using confocal microscopy in human neutrophils which phagocytized live <i>C. albicans</i>. The distribution of intracellular Dectin-1 was indicated by red arrows and ROS by green arrows. The neutrophils which phagocytized <i>C. albicans</i> spores were indicated by yellow arrows. The localization association of intracellular Dectin-1 and stimulated ROS was indicated by blue arrows in the merged fluorescence image. <b>C.</b> rhDectin-1 binding before the treatment with <i>β</i>-1,3-glucanase. <b>D.</b> rhDectin-1 binding after the treatment with <i>β</i>-1,3-glucanase. <b>C</b> and <b>D</b>. Exposure of <i>β</i>-1,3-glucan on the spores of <i>C. albicans</i> was confirmed by indirect immunofluorescence assay using hrDectin-1 and PE-anti-human Dectin-1 mAb, and <i>β</i>-1,3-glucanase digestion test. The binding of Dectin-1 on the cell wall of <i>C. albicans</i> was indicted by blue arrows in the merged image. The data represented 3 similar experiments. Scale bars, <b>A, B</b> and <b>C.</b> 10 µm; <b>D.</b> 20 µm.</p

The inhibited phagocytic efficiency of human neutrophils by blockage of Dectin-1 was compensated by exogenous MBL.

<p><b>A.</b> Abrogation effect of Dectin-1 on human neutrophils by 5 µg/mL blocking mAb was measured by flow cytometry. PE-mouse IgG2b was used as isotype control. <b>B</b> and <b>C.</b> Neutrophils containing intracellular FITC-<i>C. albicans</i> had distinctive green fluorescence, and were easily differentiated from the ones without intracellular FITC- <i>C. albicans</i>. Accordingly, the phagocytic efficiency of neutrophils was measured by flow cytometry assay after stimulation with FITC-<i>C. albicans</i> for 30 and 60 min in the presence of 5 µg/mL Dectin-1 blocking mAb and exogenous MBL at a series of concentrations of 2.5, 5 and 10 µg/mL. <b>D.</b> Bar graph depicted the phagocytic efficiency of human neutrophils at 30 or 60 min after stimulation by FITC-<i>C. albicans</i> in the presence of 5 µg/mL Dectin-1 blocking mAb and exogenous MBL at a series of concentrations of 2.5, 5 and 10 µg/mL. Data were represented as mean ± SE (n = 20). * Significant (<0.05), ** highly significant (<0.01). <b>E</b> and <b>F.</b> Linear regression analysis between the phagocytic efficiency of human neutrophils at 30 and 60 min after stimulation by FITC-<i>C. albicans</i>, and the dosage of exogenous MBL. ** Highly significant (P<0.01).</p

MBL<i>-</i>pre-incubated <i>C. albicans</i> stimulated mRNA expression of Dectin-1 in human neutrophils.

<p><b>A.</b> Products obtained by using human Dectin-1 cDNA primers were sequenced and compared to the reference sequence in NCBI. <b>B.</b> Bar graph depicted the fold expression of neutrophil Dectin-1 mRNA at indicated time points after stimulation by live or HK-<i>C. albicans</i> at a MOI of 10 which was pre-incubated with 10 µg/mL MBL for 30 min at 37°C. Data were represented as mean ± SE (n = 20). ** Highly significant (P<0.01).</p

MBL-Mediated Opsonophagocytosis of Candida albicans by Human Neutrophils Is Coupled with Intracellular Dectin-1-Triggered ROS Production

Mannan-binding lectin (MBL), a lectin homologous to C1q, greatly facilitates C3/C4-mediated opsonophagocytosis of Candida albicans (C. albicans) by human neutrophils, and has the capacity to bind to CR1 (CD35) expressed on circulating neutrophils. The intracellular pool of neutrophil Dectin-1 plays a critical role in stimulating the reactive oxygen species (ROS) generation through recognition of beta-1,3-glucan component of phagocytized zymosan or yeasts. However, little is known about whether MBL can mediate the opsonophagocytosis of Candida albicans by neutrophils independent of complement activation, and whether MBL-mediated opsonophagocytosis influence the intracellular expression of Dectin-1 and ROS production. Here we showed that the inhibited phagocytic efficiency of neutrophils as a result of blockage of Dectin-1 was compensated by exogenous MBL alone in a dose-dependent manner. Furthermore, the expressions of Dectin-1 at mRNA and intracellular protein levels were significantly up-regulated in neutrophils stimulated by MBL-pre-incubated C. albicans, while the expression of surface Dectin-1 remained almost unchanged. Nevertheless, the stimulated ROS production in neutrophils was partly and irreversibly inhibited by blockage of Dectin-1 in the presence of exogenous MBL. Confocal microscopy examination showed that intracellular Dectin-1 was recruited and co-distributed with ROS on the surface of some phagocytized yeasts. The beta-1,3-glucanase digestion test further suggested that the specific recognition and binding site of human Dectin-1 is just the beta-1,3-glucan moiety on the cell wall of C. albicans. These data demonstrate that MBL has an ability to mediate the opsonophagocytosis of Candida albicans by human neutrophils independent of complement activation, which is coupled with intracellular Dectin-1-triggered ROS production.Mannan-binding lectin (MBL), a lectin homologous to C1q, greatly facilitates C3/C4-mediated opsonophagocytosis of Candida albicans (C. albicans) by human neutrophils, and has the capacity to bind to CR1 (CD35) expressed on circulating neutrophils. The intracellular pool of neutrophil Dectin-1 plays a critical role in stimulating the reactive oxygen species (ROS) generation through recognition of beta-1,3-glucan component of phagocytized zymosan or yeasts. However, little is known about whether MBL can mediate the opsonophagocytosis of Candida albicans by neutrophils independent of complement activation, and whether MBL-mediated opsonophagocytosis influence the intracellular expression of Dectin-1 and ROS production. Here we showed that the inhibited phagocytic efficiency of neutrophils as a result of blockage of Dectin-1 was compensated by exogenous MBL alone in a dose-dependent manner. Furthermore, the expressions of Dectin-1 at mRNA and intracellular protein levels were significantly up-regulated in neutrophils stimulated by MBL-pre-incubated C. albicans, while the expression of surface Dectin-1 remained almost unchanged. Nevertheless, the stimulated ROS production in neutrophils was partly and irreversibly inhibited by blockage of Dectin-1 in the presence of exogenous MBL. Confocal microscopy examination showed that intracellular Dectin-1 was recruited and co-distributed with ROS on the surface of some phagocytized yeasts. The beta-1,3-glucanase digestion test further suggested that the specific recognition and binding site of human Dectin-1 is just the beta-1,3-glucan moiety on the cell wall of C. albicans. These data demonstrate that MBL has an ability to mediate the opsonophagocytosis of Candida albicans by human neutrophils independent of complement activation, which is coupled with intracellular Dectin-1-triggered ROS production

Fluorescence intensity of FITC-<i>C. albicans</i> was homogeneously distributed.

<p><b>A.</b> FITC-<i>C. albicans</i> was selected according to side scatter (SSC) and forward scatter (FSC). Fluorescence intensity of FITC-<i>C. albicans</i> was detected by flow cytometry. <b>B.</b> FITC-<i>C. albicans</i> was examined by Laser Confocal microscopy (10×100). The merged image (left below) showed the distribution of FITC on the <i>C. albicans</i>. Scale bar, 5 µm.</p

In vitro induced transformation of saprophytic <i>F</i>. <i>pedrosoi</i> into sclerotic cells.

<p>Optical microscope was used to characterize the morphology of saprophytic <i>F</i>. <i>pedrosoi</i> growing on PDA (A) and in-vitro transformed sclerotic cells cultured in ATCC830 medium plus PAF for 50 days (×400) (B). Scale bar = 20 μm (A and B).</p