*Contributed equally
Dendritic cell-associated C-type lectin-1 (Dectin-1), a C-type lectin receptor, serves a critical role in host antifungal immunity. However, the molecular mechanism and function of Dectin-1-mediated signaling in response to infection by the pathogenic fungus
Talaromycosis is a severe deep mycosis caused by
Innate immunity represents the first line of defense for hosts against microbes. Upon invasion, pathogens are suppressed by the host innate immune system through the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). It is well established that NOD-like receptors (NLRs), Toll-like receptors (TLRs), C-type lectin receptors (CLRs) and retinoic acid-inducible gene I-like receptors are the best characterized PRRs for sensing different types of PAMPs (
Despite Dectin-1 playing an important role in regulating host immunity and fungal infection, the activation of Dectin-1 induced by
RPMI-1640 medium, FBS, penicillin-streptomycin solution and β-mercaptoethanol were purchased from Gibco (Thermo Fisher Scientific, Inc.). TRIzol® reagent was obtained from Invitrogen (Thermo Fisher Scientific, Inc.). PMA and puromycin were purchased from Sigma-Aldrich (Merck KGaA). One Step TB Green™ Prime Script™ RT-PCR kit II was purchased from Takara Bio, Inc. RIPA lysis buffer was purchased from Beijing Solarbio Science & Technology Co., Ltd. BSA, BCA assay and blocking buffer were purchased from Beyotime Institute of Biotechnology. ECL was purchased from Bio-Rad Laboratories, Inc. Dectin-1 short hairpin (sh)RNA lentiviral particles and scramble shRNA lentiviral particles encoding a GFP sequence were constructed by Shanghai GeneChem Co., Ltd. Antibodies against Dectin-1 (cat. no. 60128), NF-κB p65 (cat. no. 8242), phosphorylated (p)-NF-κB p65 (cat. no. 3033), IκBα (cat. no. 4814S), p-IκBα (cat. no. 9246), Syk (cat. no. 2712), p-Syk (cat. no. 2710) and anti-rabbit IgG (H + L), Alexa Fluor® 555-conjugated anti-rabbit IgG (cat. no. 4413) were purchased from Cell Signaling Technology, Inc.; horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (cat. no. L3012 was purchased from Signalway Antibody LLC; HRP-conjugated goat anti-mouse IgG secondary antibody (cat. no. ab6789) was purchased from Abcam; and anti-β-actin (cat. no. 66009-1-Ig) was purchased from ProteinTech Group, Inc.
The human monocyte cell line THP-1 (The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences) was cultured in RPMI-1640 medium supplemented with 10% inactivated FBS, 0.05 mM β-mercaptoethanol and 100 U/ml penicillin-streptomycin solution in a humidified atmosphere containing 5% CO2 at 37˚C. THP-1 cells could be differentiated into macrophages by treatment with 100 ng/ml PMA at 37˚C for 48 h. The cells were then cultured in medium without PMA.
This study used a customized PCR array from CT Bioscience to analyze the expression of key genes that participated in
The design of the Dectin-1 shRNA (interference sequence, 5'-CAATTACAC TTCGACTCTCAA-3'), a scrambled shRNA (5'-TTCTCCGA ACGTGTCACGT-3') and the packaging of the lentivirus particles were performed by Shanghai GeneChem Co., Ltd. A total of 4x104 THP-1 cells were cultured in supplemented RPMI medium in 96-well plates for 24 h. Subsequently, 4 µl HitransG P (25X; Shanghai GeneChem Co., Ltd.) enhanced infection solution was added prior to cell transduction with lentiviral particles at a multiplicity of infection (MOI) of 50 (virus number/cell number =50). Cells were incubated for 12 h, and then the culture medium was replaced with fresh medium. GFP expression was observed by fluorescence microscopy 72 h after transduction. After 72 h of infection, fresh medium containing 2 µg/ml puromycin was added for 72 h to select positively stably transduced cells. Stably transduced cells were maintained in 1 µg/ml puromycin. The third passage of stable clones was collected at 9 days after transduction for reverse transcription (RT)-qPCR analysis.
THP-1 macrophages were incubated with heat-killed
THP-1 macrophages were incubated with heat-killed
Cells were cultured on glass dishes. THP-1 macrophages were incubated with heat-killed
After stimulation of THP-1 macrophages (5x105 cells/ml) with heat-killed
Experiments were conducted at least three times. Data are presented as the mean ± standard deviation. Differences among the groups were evaluated using one-way ANOVA followed by Tukey's post hoc test. Intragroup (time) and intergroup (fluences) comparisons of the Cytokine quantification were analyzed by two-way ANOVA followed by Tukey's post hoc test using SPSS version 22.0 (IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.
To study the Dectin-1 expression patterns in the antifungal immune response, THP-1 macrophages were exposed to heat-killed
To further confirm the effect of
Since the expression of inflammatory cytokines is regulated by NF-κB, the levels of TNF-α and IL-8 were finally detected using an ELISA. The interaction between
Due to the lack of effective antifungal agents, talaromycosis is well known as a severe disease that can cause disseminated infection, and treatment of this disease remains a challenge (
To investigate whether Dectin-1 recognized the spores and hyphae of
NF-κB is an important transcriptional regulator, which controls the expression of various pro-inflammatory mediators. The NF-κB family of transcription factors consists of five members, p50, p52, p65 (RelA), c-Rel and RelB. The underlying mechanism of NF-κB signaling consists of a series of positive and negative regulatory elements. Firstly, inducing stimuli initiate IKK activation leading to phosphorylation, ubiquitination and degradation of IκB proteins. IκB is an inhibitory protein that acts to prevent NF-κB migration into the nucleus, and the degradation of IκB leads to the release of NF-κB p65/p50 dimers into the nucleus (
Inflammatory cytokines play a critical role in the development of fungal infectious diseases. Monocytes/macrophages are an important part of innate immunity against fungi, and they primarily produce cytokines, such as TNF-α, IL-1, IL-6, IL-8 and granulocyte-colony stimulating factor (
In conclusion, the present study confirmed that Dectin-1 was involved in the recognition of
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
YP, YC and WS performed the experiments. YP and YC prepared the manuscript. YaW, JM, WZ, HZ and WS analyzed the data. HZ, YuW, WZ and WS were responsible for study conception and design of the study. JM and YaW contributed to literature searching and processing. WZ and WS confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
Dectin-1 expression is increased following
Knockdown of Dectin-1 inhibits cytokine release from macrophages. (A and B) THP-1 macrophages were exposed to
Schematic representation of Dectin-1 and its roles in the immune response to