Fungal infections can lead to severe clinical outcomes such as multiple organ failure and septic shock. Rapid detection of fungal infections allows clinicians to treat patients in a timely manner and ...improves clinical outcomes. Conventional detection methods include blood culture followed by plate culture and polymerase chain reaction. These methods are time-consuming and require expensive equipment, hence, they are not suitable for point-of-care and clinical settings. There is an unmet need to develop a rapid and inexpensive detection method for fungal infections such as candidemia. We developed an innovative immuno-based microfluidic device that can rapidly detect and capture Candida albicans from phosphate-buffered saline (PBS) and human whole blood. Our microchip technology showed an efficient capture of C. albicans in PBS with an efficiency of 61–78% at various concentrations ranging from 10 to 105 colony-forming units per milliliter (cfu/mL). The presented microfluidic technology will be useful to screen for various pathogens at the point-of-care and clinical settings.
No consensus exists regarding optimal dosing of norepinephrine in septic shock. We aimed to evaluate if weight-based dosing (WBD) lead to higher norepinephrine doses when achieving goal mean arterial ...pressure (MAP) than non-weight-based dosing (non-WBD). This was a retrospective cohort study conducted after standardization of norepinephrine dosing within a cardiopulmonary ICU. Patients received non-WBD prior to standardization (November 2018-October 2019) and WBD afterwards (November 2019-October 2020). The primary outcome was the norepinephrine dose needed to attain goal MAP. Secondary outcomes included time to goal MAP, duration of norepinephrine therapy, duration of mechanical ventilation, and treatment-related adverse effects. A total of 189 patients were included (WBD 97; non-WBD 92). There was a significantly lower norepinephrine dose at goal MAP (WBD 0.05, IQR 0.02, 0.07; non-WBD 0.07, IQR 0.05, 0.14;
< 0.005) and initial norepinephrine dose (WBD 0.02, IQR 0.01, 0.05; non-WBD 0.06, 0.04, 0.12;
< 0.005) in the WBD group. No difference was observed in achievement of goal MAP (WBD 73%; non-WBD 78%;
= 0.09) or time until goal MAP (WBD 18, IQR 0, 60; non-WBD 30, IQR 14, 60;
= 0.84). WBD may lead to lower norepinephrine doses. Both strategies achieved goal MAP with no significant difference in time to goal.
Autophagy has been postulated to play role in mammalian host defense against fungal pathogens, although the molecular details remain unclear. Here, we show that primary macrophages deficient in the ...autophagic factor LC3 demonstrate diminished fungicidal activity but increased cytokine production in response to Candida albicans stimulation. LC3 recruitment to fungal phagosomes requires activation of the fungal pattern receptor dectin-1. LC3 recruitment to the phagosome also requires Syk signaling but is independent of all activity by Toll-like receptors and does not require the presence of the adaptor protein Card9. We further demonstrate that reactive oxygen species generation by NADPH oxidase is required for LC3 recruitment to the fungal phagosome. These observations directly link LC3 to the inflammatory pathway against C. albicans in macrophages.
Dectin-1 and TLR9 play distinct roles in the recognition and induction of innate immune responses to Aspergillus fumigatus and Candida albicans. Dectin-1 is a receptor for the major fungal cell wall ...carbohydrate β-1,3 glucan that induces inflammatory cytokines and controls phagosomal maturation through spleen tyrosine kinase activation. TLR9 is an endosomal TLR that also modulates the inflammatory cytokine response to fungal pathogens. In this study, we demonstrate that β-1,3 glucan beads are sufficient to induce dynamic redistribution and accumulation of cleaved TLR9 to phagosomes. Trafficking of TLR9 to A. fumigatus and C. albicans phagosomes requires Dectin-1 recognition. Inhibition of phagosomal acidification blocks TLR9 accumulation on phagosomes containing β-1,3 glucan beads. Dectin-1-mediated spleen tyrosine kinase activation is required for TLR9 trafficking to β-1,3 glucan-, A. fumigatus-, and C. albicans-containing phagosomes. In addition, Dectin-1 regulates TLR9-dependent gene expression. Collectively, our study demonstrates that recognition of β-1,3 glucan by Dectin-1 triggers TLR9 trafficking to β-1,3 glucan-containing phagosomes, which may be critical in coordinating innate antifungal defense.
Elimination of fungal pathogens by phagocytes requires phagosome maturation, a process that involves the recruitment and fusion of intracellular proteins. The role of Dectin-1, a β-1,3-glucan ...receptor, critical for fungal recognition and triggering of Th17 responses, to phagosomal maturation has not been defined. We show that GFP-Dectin-1 translocates to the fungal phagosome, but its signal decays after 2 h. Inhibition of acidification results in retention of GFP-Dectin-1 to phagosome membranes highlighting the requirement for an acidic pH. Following β-1,3-glucan recognition, GFP-Dectin-1 undergoes tyrosine phosphorylation by Src kinases with subsequent Syk activation. Our results demonstrate that Syk is activated independently of intraphagosomal pH. Inhibition of Src or Syk results in prolonged retention of GFP-Dectin-1 to the phagosome signifying a link between Syk and intraphagosomal pH. β-1,3-glucan phagosomes expressing a signaling incompetent Dectin-1 failed to mature as demonstrated by prolonged Dectin-1 retention, presence of Rab5B, failure to acquire LAMP-1 and inability to acidify. Phagosomes containing Candida albicans also require Dectin-1-dependent Syk activation for phagosomal maturation. Taken together, these results support a model where Dectin-1 not only controls internalization of β-1,3-glucan containing cargo and triggers proinflammatory cytokines, but also acts as a master regulator for subsequent phagolysosomal maturation through Syk activation.
Background: Dectin-1 is able to recognize and phagocytose the fungal carbohydrate, β-1,3-glucan, but its contribution to phagosomal maturation has not been explored.
Results: Dectin-1-dependent Syk activation promotes phagolysosomal fusion and acidification.
Conclusion: Dectin-1-dependent Syk-activation permits egress of early phagosomes to mature phagolysosomes.
Significance: The surface recognition receptor, Dectin-1 shapes anti-fungal responses by controlling fungal phagosome maturation.
Macrophages play a critical role in the elimination of fungal pathogens. They are sensed
cell surface pattern-recognition receptors and are phagocytosed into newly formed organelles called ...phagosomes. Phagosomes mature through the recruitment of proteins and lysosomes, resulting in addition of proteolytic enzymes and acidification of the microenvironment. Our earlier studies demonstrated an essential role of Dectin-1-dependent activation of spleen tyrosine kinase (Syk) in the maturation of fungal containing phagosomes. The absence of Syk activity interrupted phago-lysosomal fusion resulting in arrest at an early phagosome stage. In this study, we sought to define the contribution of Syk to the control of phagocytosed live
in primary macrophages. To accurately measure intracellular yeast division, we designed a carboxyfluorescein succinimidyl ester (CFSE) yeast division assay in which bright fluorescent parent cells give rise to dim daughter cells. The CFSE-labeling of
did not affect the growth rate of the yeast. Following incubation with macrophages, internalized CFSE-labeled
were retrieved by cellular lysis, tagged using ConA-647, and the amount of residual CFSE fluorescence was assessed by flow cytometry.
remained undivided (CFSE bright) for up to 18 h in co-culture with primary macrophages. Treatment of macrophages with R406, a specific Syk inhibitor, resulted in loss of intracellular control of
with initiation of division within 4 h. Delayed Syk inhibition after 8 h was less effective indicating that Syk is critically required at early stages of macrophage-fungal interaction. In conclusion, we demonstrate a new method of tracking division of
using CFSE labeling. Our results suggest that early Syk activation is essential for macrophage control of phagocytosed
.
CD82 controls CpG‐dependent TLR9 signaling Khan, Nida S.; Lukason, Daniel P.; Feliu, Marianela ...
The FASEB journal,
November 2019, 2019-11-00, 20191101, Letnik:
33, Številka:
11
Journal Article
Recenzirano
Odprti dostop
The tetraspanin CD82 is a potent suppressor of tumor metastasis and regulates several processes including signal transduction, cell adhesion, motility, and aggregation. However, the mechanisms by ...which CD82 participates in innate immunity are unknown. We report that CD82 is a key regulator of TLR9 trafficking and signaling. TLR9 recognizes unmethylated cytosine‐phosphate‐guanine (CpG) motifs present in viral, bacterial, and fungal DNA. We demonstrate that TLR9 and CD82 associate in macrophages, which occurs in the endoplasmic reticulum (ER) and post‐ER. Moreover, CD82 is essential for TLR9‐dependent myddosome formation in response to CpG stimulation. Finally, CD82 modulates TLR9‐dependent NF‐αB nuclear translocation, which is critical for inflammatory cytokine production. To our knowledge, this is the first time a tetraspanin has been implicated as a key regulator of TLR signaling. Collectively, our study demonstrates that CD82 is a specific regulator of TLR9 signaling, which may be critical in cancer immunotherapy approaches and coordinating the innate immune response to pathogens.—Khan, N. S., Lukason, D. P., Feliu, M., Ward, R. A., Lord, A. K., Reedy, J. L., Ramirez‐Ortiz, Z. G., Tam, J. M., Kasperkovitz, P. V., Negoro, P. E., Vyas, T. D., Xu, S., Brinkmann, M. M., Acharaya, M., Artavanis‐Tsakonas, K., Frickel, E.‐M., Becker, C. E., Dagher, Z., Kim, Y.‐M., Latz, E., Ploegh, H. L., Mansour, M. K., Miranti, C. K., Levitz, S. M., Vyas, J. M. CD82 controls CpG‐dependent TLR9 signaling. FASEB J. 33, 12500–12514 (2019). www.fasebj.org
Tetraspanins are a family of proteins possessing four transmembrane domains that help in lateral organization of plasma membrane proteins. These proteins interact with each other as well as other ...receptors and signaling proteins, resulting in functional complexes called "tetraspanin microdomains." Tetraspanins, including CD82, play an essential role in the pathogenesis of fungal infections. Dectin-1, a receptor for the fungal cell wall carbohydrate β-1,3-glucan, is vital to host defense against fungal infections. The current study identifies a novel association between tetraspanin CD82 and Dectin-1 on the plasma membrane of
-containing phagosomes independent of phagocytic ability. Deletion of CD82 in mice resulted in diminished fungicidal activity, increased
viability within macrophages, and decreased cytokine production (TNF-α, IL-1β) at both mRNA and protein level in macrophages. Additionally, CD82 organized Dectin-1 clustering in the phagocytic cup. Deletion of CD82 modulates Dectin-1 signaling, resulting in a reduction of Src and Syk phosphorylation and reactive oxygen species production. CD82 knockout mice were more susceptible to
as compared with wild-type mice. Furthermore, patient
-induced cytokine production was influenced by two human CD82 single nucleotide polymorphisms, whereas an additional CD82 single nucleotide polymorphism increased the risk for candidemia independent of cytokine production. Together, these data demonstrate that CD82 organizes the proper assembly of Dectin-1 signaling machinery in response to
.
Invasive fungal infections constitute a lethal threat, with patient mortality as high as 90%. The incidence of invasive fungal infections is increasing, especially in the setting of patients ...receiving immunomodulatory agents, chemotherapy, or immunosuppressive medications following solid-organ or bone marrow transplantation. In addition, inhibitors of spleen tyrosine kinase (Syk) have been recently developed for the treatment of patients with refractory autoimmune and hematologic indications. Neutrophils are the initial innate cellular responders to many types of pathogens, including invasive fungi. A central process governing neutrophil recognition of fungi is through lectin binding receptors, many of which rely on Syk for cellular activation. We previously demonstrated that Syk activation is essential for cellular activation, phagosomal maturation, and elimination of phagocytosed fungal pathogens in macrophages. Here, we used combined genetic and chemical inhibitor approaches to evaluate the importance of Syk in the response of neutrophils to
species. We took advantage of a Cas9-expressing neutrophil progenitor cell line to generate isogenic wild-type and Syk-deficient neutrophils. Syk-deficient neutrophils are unable to control the human pathogens
,
, and
Neutrophil responses to
species, including the production of reactive oxygen species and of cytokines such as tumor necrosis factor alpha (TNF-α), the formation of neutrophil extracellular traps (NETs), phagocytosis, and neutrophil swarming, appear to be critically dependent on Syk. These results demonstrate an essential role for Syk in neutrophil responses to
species and raise concern for increased fungal infections with the development of Syk-modulating therapeutics.
Neutrophils are recognized to represent significant immune cell mediators for the clearance and elimination of the human-pathogenic fungal pathogen
The sensing of fungi by innate cells is performed, in part, through lectin receptor recognition of cell wall components and downstream cellular activation by signaling components, including spleen tyrosine kinase (Syk). While the essential role of Syk in macrophages and dendritic cells is clear, there remains uncertainty with respect to its contribution in neutrophils. In this study, we demonstrated that Syk is critical for multiple cellular functions in neutrophils responding to major human-pathogenic
species. These data not only demonstrate the vital nature of Syk with respect to the control of fungi by neutrophils but also warn of the potential infectious complications arising from the recent clinical development of novel Syk inhibitors for hematologic and autoimmune disorders.
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