text
string | predicted_class
string | confidence
float16 |
|---|---|---|
CaLC3672/CaLC3673: To introduce a mutant allele of CAS5 carrying S462A, S464A, S472A, and S476A mutations, the CAS5S 462A/S464A/S472A/S476A -HA-HIS1 cassette was released from pLC858 with SacII and transformed into CaLC2034. Transformants prototrophic for histidine were PCR tested as described for CaLC3693. The absence of an untagged wildtype CAS5 allele was verified with oLC3052 in combination with oLC2164. The S462A/S464A/S472A/S476A mutations were sequence verified with oLC3371.
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study
| 100.0 |
CaLC4471: To C terminally TAP tag Swi4 in a mutant heterozygous for SWI4, the TAP-ARG4 cassette was PCR amplified from pLC57362 using primers oLC3424 in combination with oLC3425 and transformed into CaLC3151. Transformants prototrophic for arginine were PCR tested with oLC3426 in combination with oLC3427.
|
study
| 99.94 |
CaLC4499: To C terminally TAP tag Swi6 in a mutant heterozygous for SWI6, the TAP-ARG4 cassette was PCR amplified from pLC57362 using primers oLC3428 in combination with oLC3429 and transformed into SWI6/swi6::FRT. Transformants prototrophic for arginine were PCR tested with oLC3430 in combination with oLC1593 and oLC3430 in combination with oLC3431.
|
study
| 99.94 |
CaLC4705: To C terminally RFP tag Hhf1 in SN95, the RFP-NAT cassette was PCR amplified from pLC447 using primers oLC4752 in combination with oLC4753 and transformed into CaLC239. Correct integration at the C terminus of HHF1 was verified by amplifying across both junctions using primer pairs oLC4417 in combination with oLC4754 and oLC274 in combination with oLC4755. To C terminally GFP tag Dad2, the GFP-HIS cassette was PCR amplified from pLC38363 using primers oLC4748 in combination with oLC4749 and transformed into HHF1-RFP/HHF1. Correct integration at the C terminus of DAD2 was verified by amplifying across both junctions using primer pairs oLC600 in combination with oLC4750 and oLC1645 in combination with oLC4751.
|
study
| 99.94 |
CaLC4707: To C terminally RFP tag Hhf1 in a cas5Δ/cas5Δ mutant, the RFP-NAT cassette was PCR amplified from pLC447 as described for CaLC4705 and transformed into CaLC2056. Correct integration at the C terminus of HHF1 was verified as described for CaLC4705. To C terminally GFP tag Dad2, the GFP-HIS cassette was PCR amplified from pLC38363 as described for CaLC4705 and transformed into cas5Δ/cas5Δ HHF1-RFP/HHF1. Correct integration at the C terminus of DAD2 was verified by as described for CaLC4705.
|
study
| 99.94 |
Cloning procedures were performed following standard protocols. Plasmids used in this study are listed in Supplementary Table 2. The absence of nonsynonymous mutations in plasmids was verified by sequencing. Primers used in this study are listed in Supplementary Table 3. Plasmid construction is described in detail below.
|
study
| 86.25 |
pLC447: This is a construct to tag proteins with RFP at the C terminus. CaCherry/RFP was PCR amplified from pLC43564 with oLC841 in combination with oLC842 and digested with BsrGI. This was cloned into pLC389 at BsrGI. Directionality of the insert was verified by PCR with pLC849 in combination with oLC842. The construct was sequence verified with oLC849 and oLC842.
|
study
| 99.9 |
pLC790: This is a construct to C terminally HA-tag Cas5 in C. albicans. The CAS5 DNA-binding domain, HA, and HIS marker were amplified from CaLC3113 genomic DNA with primers oLC3092 in combination with oLC3052 and cloned into pLC49 at the ApaI site. The ligation mixture was transformed into TOP10 cells. Cells were plated on LB + Amp + NAT. Correct integration of the cassette in pLC49 was sequence verified with oLC2029. This construct can be liberated by digestion with ApaI and transformed into C. albicans.
|
study
| 100.0 |
pLC791: This plasmid is based on pLC790 but harbors a phosphomimetic mutation in CAS5 (S769E). This mutation was introduced by site-directed mutagenesis with primers oLC2986 and oLC2987. The clone was sequence verified with oLC2029. This construct can be liberated by digestion with ApaI and transformed into C. albicans.
|
study
| 99.94 |
pLC800: This plasmid is based on pLC790 but harbors a phosphomimetic mutation in CAS5 (S772E). This mutation was introduced by site-directed mutagenesis with primers oLC2990 and oLC2991. The plasmid was sequence verified with oLC2029. This construct can be liberated by digestion with ApaI and transformed into C. albicans.
|
study
| 99.94 |
pLC818: This is a construct to C terminally HA-tag Cas5 in C. albicans. The CAS5 ORF, HA, and HIS1 were amplified from CaLC3113 genomic DNA with primers oLC3365 in combination with oLC3366 and cloned into pLC49 at the SacII site. The ligation mixture was transformed into TOP10 cells. Cells were plated on LB + Amp + NAT. Correct integration of the cassette in pLC49 was sequence verified with oLC2029, oLC244, and oLC3371. This construct can be liberated by digestion with SacII and transformed into C. albicans.
|
study
| 100.0 |
pLC828: This plasmid is based on pLC818 but harbors a phosphomimetic mutation in CAS5 (S472E/S476E). This mutation was introduced by site-directed mutagenesis with primers oLC3410 and oLC3411. The plasmid was sequence verified with oLC3371. This construct can be liberated by digestion with SacII and transformed into C. albicans.
|
study
| 99.94 |
pLC857: This plasmid is based on pLC828 but harbors additional phosphomimetic mutations in CAS5 (S462E/S464E). This mutation was introduced by site-directed mutagenesis with primers oLC3416 and oLC3417. The plasmid was sequence verified with oLC3371. This construct can be liberated by digestion with SacII and transformed into C. albicans.
|
study
| 99.94 |
pLC833: This plasmid is based on pLC818 but harbors a phosphodeficient mutation in CAS5 (S472A/S476A). This mutation was introduced by site-directed mutagenesis with primers oLC3412 and oLC3413. The clone was sequence verified with oLC3371. This construct can be liberated by digestion with SacII and transformed into C. albicans.
|
study
| 99.94 |
pLC858: This plasmid is based on pLC833 but harbors additional phosphodeficient mutations in CAS5 (S462A/S464A). This mutation was introduced by site-directed mutagenesis with primers oLC3418 and oLC3419. The plasmid was sequence verified with oLC3371. This construct can be liberated by digestion with SacII and transformed into C. albicans.
|
study
| 99.94 |
All cultures were grown shaking at 200 rpm. To assess the effects of caspofungin and calcofluor white treatment on the expression of Cas5 and Cas5-dependent of transcripts, overnight cultures were diluted to OD600 of 0.15 in YPD for 3 h, and either untreated or treated with 125 ng/ml of caspofungin or 100 µg/ml of calcofluor white for 1 or 2 h, as specified. To deplete GLC7, strains were grown overnight at 30 °C in YPD. Stationary phase cultures were split, adjusted to an OD600 of 0.15, at which point one culture was treated with 0.02 µg/ml of doxycycline, whereas the other was left untreated. Cells were grown for 16 h and were split again, adjusted to an OD600 of 0.15 and grown for 3 h. One culture was subsequently treated with 125 ng/ml of caspofungin for 1 or 2 h as specified, whereas the other was left untreated.
|
study
| 100.0 |
Chromatin preparation and immunoprecipitation against PolII using 2 µl of 8WG16 antibody were performed as described previously65. Multiplex sequencing libraries were constructed according to Wong et al.66 ChIP-Seq libraries were assessed by Bioanalyzer DNA High Sensitivity Assay and quantified using real-time PCR before sequencing on the Illumina HiSeq2500 platform. Raw reads were mapped to the C. albicans reference genome. Mapped reads on gene bodies were counted, normalized to total number of mapped reads and expressed as “Normalized PolII ChIP-Seq signal”. Differentially bound genes are defined by greater than 1.5-fold difference in PolII ChIP-Seq signals between any two conditions analyzed. Non-expressed genes or lowly expressed genes (e.g., genes with PolII ChIP-Seq signals lower than 10) under both conditions analyzed were removed from downstream analysis. Pathway analysis was carried out using the GO program on the Candida Genome Database (http://www.candidagenome.org/cgi-bin/GO/goTermFinder) and the KEGG mapper (http://www.genome.jp/kegg/tool/map_pathway2.html).
|
study
| 100.0 |
Antifungal susceptibility was measured in flat bottom, 96-well microtitre plates (Sarstedt #83.3924) using a broth microdilution protocol described in ref. 67. In brief, minimum inhibitory concentration (MIC) assays were set up in twofold serial dilutions of caspofungin or calcofluor white in a final volume of 200 µl per well. Caspofungin gradients were diluted either from 125 ng/ml down to 0.12 ng/ml or 2000 to 3.9 ng/ml. Calcofluor white gradients were diluted from 250 µg/ml down to 0.24 µg/ml. Where applicable, doxycycline was added to a final concentration of 0.1 µg/ml. Cell densities of overnight cultures were determined and dilutions were prepared such that ~103 cells were inoculated into each well. Plates were incubated in the dark at 30 °C for 48 h, at which point the absorbance was determined at 600 nm using a spectrophotometer (Molecular Devices) and corrected for background from the corresponding medium. Each strain was tested in duplicate in three biological replicates. MIC data was quantitatively displayed with color using the program Java TreeView 1.1.3 (http://jtreeview.sourceforge.net).
|
study
| 100.0 |
Yeast strains were cultured overnight in 4 ml YPD at 30 °C. Cells were subcultured for 4–5 h and isolated during exponential growth. Overall, 400 μl of cells were pelleted and fixed overnight in 70% ethanol at 4 °C. The fixed cells were washed twice with 1 ml of sodium citrate (50 mM) and resuspended in 500 μl sodium citrate and 12 μl of RNaseA (25 mg/ml). The cells were incubated at 37 °C for 4 h. Then 17 μl of propidium iodide was added to the RNase-treated cells and incubated overnight at 37°C. The cells were vortexed and sonicated for 5 s at level 15 of the Sonic Desmembrator (Fisher Scientific), and filtered immediately before analysis using a BD Falcon tube with a 35 μm nylon mesh cap (Cat. No. 352235). Ploidy was determined using a Yetti flow cytometer, and 50,000–100,000 cells were analyzed per strain.
|
study
| 99.94 |
Cultures were grown to mid-exponential phase and harvested by centrifugation at 3000 rpm for 5 min. The cells were washed with sterile H2O and resuspended in 1 mL of lysis buffer (20 mM Tris pH 7.5, 100 mM KCl, 5 mM MgCl, and 20% glycerol), with one protease inhibitor cocktail per 50 ml (complete, EDTA-free tablet, Roche Diagnostics, Indianapolis, IN, USA) and 1 mM PMSF (EMD Chemicals, Gibbstown, NJ, USA). Cells were lysed by bead beating twice for 4 min with 7 min on ice between cycles. Lysates were recovered by piercing a hole in the bottom of each tube, placing each tube in a 14 ml conical tube, and centrifuging at 1300×g for three 5-min cycles, recovering the supernatant after each stage. The combined lysate was cleared by centrifugation at 21,000×g for 10 min at 4 °C. The protein concentrations were determined using the Bradford assay68. Anti-HA immunoprecipitations were done using Pierce HA-Tag IP/Co-IP kit (Thermo-Fisher Scientific, PI23610), as per manufacturer’s instructions.
|
study
| 99.94 |
Protein was extracted by pelleting cells at OD600 of 0.8, with the pellet being resuspended in 2× sample buffer (one-third volume of 6× sample buffer containing 0.35 M Tris-HCl, 10% (w/w) SDS, 36% glycerol, 5% β-mercaptoethanol, and 0.012% bromophenol blue). The samples were boiled for 5 min at 95 °C. The cell debris was pelleted and the supernatant was separated on a 6% SDS-PAGE gel to observe changes in Cas5 mobility. Separated proteins were electrotransferred to PVDF membrane (Bio-Rad Laboratories) and blocked with 5% skim milk in phosphate-buffered saline (PBS) with 0.2% Tween-20 at room temperature for 1 h. Blots were hybridized with antibody against the HA epitope (1:5000 dilution; Roche Diagnostics), p44/42 (1:2000, Cell Signaling), PSTAIRE (1:5000, Santa Cruz Biotechnology), TAP (1:5000; Open Biosystems), Hsp90 (1:10,000; generously provided by Brian Larsen, Des Moines University) or α-β-actin (1:5000; Santa Cruz, sc-47778) overnight at 4 °C. Blots were washed with PBS with 0.1% Tween-20 and subsequently hybridized with FITC-conjugated secondary antibody diluted 1:5000 in the block solution for 45 min at room temperature. Signals were detected using an ECL western blotting kit as per the manufacturer’s instructions (Pierce).
|
study
| 99.94 |
C. albicans Cas5-HA cells were harvested from log phase cultures in YPD (+/−caspofungin) with lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7, 15 mM EDTA, 1% Triton 100-X, 10% glycerol) plus phosphatase inhibitors (Sigma, P5726 and P0044) and protease inhibitors (Roche complete Mini Protease Inhibitor Cocktail Tablets 04693124001) using glass beads and bead beating 10 × 20 s with 1 min ice in between. Cell extracts were precipitated with methanol–chloroform and resuspended in rehydration buffer (Bio-Rad Cat#163-2105). Approximately 30 μg protein was loaded onto 7 cm IPG strips (pH 3-10, Bio-Rad Cat#163-2000) and resolved using Bio-Rad Protean i12 IEF System. The IPG strips were then resolved using 10% SDS-PAGE gels and processed for western blotting following the procedures described above for one-dimensional western blot analysis.
|
study
| 99.94 |
To prepare samples for RNA extraction, 10 ml of subculture was harvested by centrifugation at 1300×g for 5 min. The pellet was flash-frozen and stored at −80 °C overnight. RNA was isolated using the QIAGEN RNeasy kit and cDNA was generated using the AffinityScript cDNA synthesis kit (Stratagene). qRT-PCR was carried out using the Fast SYBR Green Master Mix (Thermo-Fisher Scientific) in 384-well plates with the following cycle conditions: 95 °C for 10 min, repeat 95 °C for 10 s, 60 °C for 30 s for 40 cycles. The melt curve was completed with the following cycle conditions: 95 °C for 10 s and 65 °C for 5 s with an increase of 0.5 °C per cycle up to 95 °C. All reactions were done in triplicate. Data were analyzed in the Bio-Rad CFX manager 3.1. Data was plotted using GraphPad Prism.
|
study
| 99.94 |
For affinity purification (AP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, clarified whole-cell lysates (10 mg protein per sample) were extracted with immobilized anti-HA beads (Pierce HA-Tag IP/Co-IP kit, Thermo-Fisher Scientific, PI23610). After a 3-h incubation, the beads were washed three times with lysis buffer and twice with HPLC water. The proteins bound on beads were eluted with 150 µl of 0.15% trifluoroacetic acid (TFA), neutralized with 100 mM NH4HCO3, and digested with trypsin. The tryptic peptides were purified by 200 µl C18 stage tips (Thermo Scientific, Rockford, IL, USA) and analyzed by Q-Exactive LC-MS/MS69. The tryptic peptides from anti-HA IP complexes were separated on a 50-cm Easy-Spray column with a 75-µm inner diameter packed with 2 µm C18 resin (Thermo Scientific, Odense, Denmark). The peptides were eluted over 120 min (250 nl/min) using a 0 to 40% acetonitrile gradient in 0.1% formic acid with an EASY nLC 1000 chromatography system operating at 50 °C (Thermo-Fisher Scientific). The LC was coupled to a Q-Exactive mass spectrometer70 by using a nano-ESI source (Thermo-Fisher Scientific). Mass spectra were acquired in a data-dependent mode with an automatic switch between a full scan and up to 10 data-dependent MS/MS scans. Target value for the full scan MS spectra was 1e6 with a maximum injection time of 120 ms and a resolution of 70,000 at m/z 400. The ion target value for MS/MS was set to 1,000,000 with a maximum injection time of 120 ms and a resolution of 17,500 at m/z 400. The first mass for the MS/MS was set to 140 m/z and the normalized collision energy was set to 27. Unassigned, as well as charge states 1 and >5 were ignored for MS/MS selection. Repeat sequencing of peptides was kept to a minimum by dynamic exclusion of sequenced peptides for 12 s71.
|
study
| 100.0 |
Acquired raw files were analyzed by using MaxQuant software72 (version 1.3.0.5) for quantification, and X! Tandem (The GPM, thegpm.org; version CYCLONE; 2010.12.01.1) and Scaffold (version Scaffold_3.4.3, Proteome Software, Portland, OR, USA) for further validation. The default search parameters were used as described by Deeb et al.71. The search included cysteine carbamidomethylation as a fixed modification, as well as N-terminal acetylation, methionine oxidation, and phospho-serine, -threonine, and -tyrosine as variable modifications. Localization probability for phosphorylation sites was required to exceed 75%. The second peptide identification option in Andromeda was enabled. For statistical evaluation of the data obtained, the posterior error probability and false discovery rate were used. The false discovery rate was determined by searching a reverse database. A false discovery rate of 0.01 for proteins and peptides was permitted. Two miscleavages were allowed, and a minimum of seven amino acids per identified peptide were required. Peptide identification was based on a search with an initial mass deviation of the precursor ion of up to 6 ppm, and the allowed fragment mass deviation was set to 20 ppm. To match identifications across different replicates and adjacent fractions, the “match between runs” option in MaxQuant was enabled within a time window of 2 min.
|
study
| 100.0 |
Cultures were grown to mid-exponential phase and 1 ml of the cells was centrifuged at 14,000 rpm for 1 min. The supernatant was removed and the cells were washed with PBS. To stain for chitin, calcofluor white was added to a final concentration of 1 µg/ml in a final volume of 100 µl. The cells were incubated in the dark for 15 min and were gently vortexted every 3–4 min. Cells were washed with PBS and 2 µl of the cells were deposited on a cover slide. To stain for nuclei, the cells were heat fixed on the slide for 1 min at 75 °C and 0.5 µl of 1 mg/ml DAPI was added. All imaging was performed on a Zeiss Imager M1 upright microscope and AxioCam Mrm with AxioVision 4.7 software. For fluorescence microscopy, an X-cite series 120 light source with ET green fluorescent protein (GFP), 4′,6-diamidino-2-phenylindole (DAPI) hybrid, and ET HQ tetramethylrhodamine isothiocyanate (TRITC)/DsRED filter sets from Chroma Technology (Bellows Falls, VT, USA) was used. Calcofluor white and DAPI were viewed under the DAPI hybrid filter and HA-tagged Cas5 was viewed under the Texas Red filter. To facilitate nuclei counting, 12 Z stacks were taken for each image, where each slice is 0.3 μm.
|
study
| 99.94 |
To obtain cells for immunofluorescence, 5–10 ml of subculture was grown to mid-exponential phase and fixed with 5% formaldehyde for 3–4 h at 30 °C. Cells were harvested at 1300×g for 5 min and washed once with 5 ml S-Buffer (50 mM HEPES pH 7.5, 1.2 M Sorbitol) before being resuspended in 1 ml S-Buffer. To induce spheroplast formation, 10 µl of 1 M DTT, 30 µl of glusulase, and 40 µl of 2.5 mg/ml zymolase were added to the cells and the mix was incubated for 30 min at 37 °C. The extent of spheroplasting was monitored under the microscope. The poly lysine (Lys) coated slides were prepared by adding 15 µl of 0.1% poly Lys per slide well and set aside for 15 min. Wells were washed three times with PBS. The fixed cells were centrifuged for 1 min at 5000 rpm and gently resuspended in 1 ml S-buffer. Following the addition of 0.1% Triton X-100, the mix was incubated for 5 min on rocker. The cells were centrifuged for 1 min at 5000 rpm and resuspended in 1 ml S-Buffer. To adhere the cells, 20 µl of cell suspension was added on to poly Lys-coated well and incubated it for 15 min. The cells were washed three times with PBS-T and the wells were blocked with 20 µl PBS/BSA for 5–10 min. Anti-HA antibody diluted 1:300 in 20 µl was added and incubated for 3–4 h or overnight in a humid chamber. The cells were washed four times in PBS-T. Anti-mouse IgG-Cy3 diluted 1:500 in PBS-T was added to the wells and incubated for 1–2 h. The cells were washed four times with PBS-T and once with only PBS. Overall, 20 µl of 1 mg/ml DAPI diluted 1:1000 in PBS was added to the wells and incubated for 5 min. The cells were washed four times with PBS. The cells were left to dry in the dark for 30 min at room temperature. Mounting medium was added and cells were viewed under the microscope.
|
study
| 99.94 |
The ChIP-seq data have been deposited in the NCBI Sequence Read Archive with accession code SRP106998. The authors declare that all other data supporting the findings of the study are available in this article and its Supplementary Information files, or from the corresponding author upon request.
|
other
| 99.94 |
The human skin is the largest organ covering the entire body and is made up of three layers, the epidermis, dermis, and subcutaneous layer . The epidermis is distinguished by five layers—the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and the stratum basale (stratum germinativum), from its exterior to the interior. The basal cells in the stratum basale divide continuously, and move toward the stratum corneum . Besides the keratinocytes, the epidermis contains other cells, such as Merkel cells, Langerhans cells, and melanocytes. The dermis is made up of connective tissue and it contains some extracellular matrix components, such as glycosaminoglycan and hyaluronic acid. The dermis possesses sweat glands, sebaceous glands , hair roots, nerves, lymph, and blood vessels. It contains three major cells—the fibroblasts, mast cells, and macrophages. There is a subcutaneous layer under the dermis containing several adipose cells that store fat and maintain body temperature . The skin has certain physiological functions, including protection from pathogens and the external environment, perception of pain or sentience, synthesis of vitamin D, temperature adjustment, absorption, and water resistance .
|
review
| 99.25 |
Paracrine factors connect between keratinocytes, fibroblasts, and melanocytes within the skin and play a vital role in ultraviolet (UV)-induced pigmentation and melanocyte activity. More recently, dermal fibroblasts were demonstrated to regulate cutaneous melanin production through secrevarious cytokines. In dark human skin, neuregulin (NRG)-1 is highly expressed by fibroblasts, suggesting its potential role in constitutive human skin color regulation. Human palmoplantar area is thicker and lighter in color than the nonpalmoplantar area. It has been demonstrated that fibroblasts produce abundant Dikkopf-1 (DKK-1) in the palms and soles, and then suppress the growth and function of melanocytes by arresting the Wnt-signaling pathway . In addition, fibroblasts also secrete numerous melanogenic factors, such as stem cell factor (SCF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), corticotropin-releasing hormone (CRH), endothelin-1 (ET-1), interferon-γ (IFN-γ), and interleukin-1 (IL-1) . Several studies have suggested dermal fibroblasts play a central role in regulating melanocyte functions and influencing human skin pigmentation and constitutive color regulation.
|
review
| 99.75 |
The stratum basale layer of the epidermis, hair, and iris contain a type of cells with the ability to produce melanin for the human body, which are known as melanocytes. Melanocytes are derived from embryonic cells and neural crest cells (NCC), and undergo several life cycles involving melanocyte differentiation from NCC into melanoblasts, the precursor cells. Melanocytes are reported to migrations and proliferations to the target site, differentiations and maturations into melanocytes, transportations and releases of melanosomes filled with melanin to keratinocytes, and cell death . The skin suffers some stresses such as inflammation and free radical accumulation, especially the UV effect on melanin production for defense against injury from the external or internal environment . Melanogenesis is a complex mechanism and process involving intercommunication between melanocytes, keratinocytes, and fibroblasts, thereby regulating signal transduction by secreting paracrine factors and cytokines. There are numerous paracrine factors which regulate melanin production, such as the proopiomelanocortin (POMC)-derived hormone, [(α-MSH and adrenocorticotropic hormone (ACTH)], ET-1, HGF, CRH, nitric oxide, and some inflammatory cytokines (IL-1β, -6, and -10, and TNF-α) . Most paracrine factors modulate melanogenesis by upregulating or downregulating the expression of the microphthalmia-associated transcription factor (MITF), which ultimately is the primary transcription factor for tyrosinase, tyrosinase-related protein-1 (TRP-1), and dopachrome tautomerase (tyrosinase related protein-2, TRP-2). Melanin synthesis is accompanied by melanosome maturation, the special cellular organelles in melanocytes. Tyrosinase plays a role as a central and rate-limiting enzyme because it initiates the reaction of tyrosine hydroxylation to L-3,4-dihydroxyphenylalanine (L-DOPA) and oxidization into DOPA quinone. In the presence of thiols such as cysteine or glutathione, DOPA quinone reacts with it and generates cysteinyl-DOPA, turning it into yellow-red pheomelanin. On the other hand, spontaneous cyclization of DOPA quinone to DOPA chrome occurs, followed by a spontaneous loss of carboxylic acid to form 5,6-dihydroxyindole (DHI). The dark-brown eumelanin is converted into indole-5,6-quinone by DHI oxidization via TRP-1 and polymerization. If the DOPA chrome is catalyzed by TRP-2 to DHI-2-carboxylic acid and then oxidized to form indole-5,6-quinone-2-carboxylic acid, its polymerization results in lighter brown eumelanin . The final step of skin pigmentation is the transport of mature melanosomes from melanocytes along with cytoskeletal elements to dendrites, followed by internalization by adjacent keratinocytes contacting with melanocytes , and 30–40 associated keratinocytes. The mature melanosomes are transported to the adjacent .
|
review
| 98.2 |
SCF is a paracrine cytokine produced by fibroblasts, keratinocytes, and endothelial cells . SCF and its receptor c-kit play an essential role in hemopoiesis and maintaining hematopoietic stem cell survival, mast cell stimulation, mediating the function of the pancreas, and melanogenesis . A mutation in SCF could cause anemia, lack of mast cells, immaturity and loss of functions of pancreatic endocrine cells, and disappearance of pigmentation, as well as the absence of melanocytes . c-Kit is a receptor tyrosine kinase expressed on melanocytes. It has two domains containing a glycosylated extracellular ligand-binding domain, comprising five Ig-like domains, and a cytoplasmic region, which comprises the protein tyrosine kinase domain. At the ectodomain, five Ig-like domains are separated from the first three domains, constituting a ligand-binding area, and two other domains act on monomerization or dimerization of c-kit. Another part, the phosphorylation site, is situated in the intracellular region, consisting of the juxtamembrane area and a large kinase-insert region . Binding of SCF to c-kit stimulates dimerization and initiation of catalytic activities of autophosphorylation and tyrosine kinase to complete its signal transmission. Y721 phosphorylation of c-kit is involved in the phosphoinositide 3-kinase (PI3K) pathway. PI3K does not only regulate cell survival but also cause pigmentation by increasing the serine/threonine-specific protein kinase AKT activity, which can produce phosphorylate glycogen synthase kinase 3β (GSK-3β) and lead to β-catenin accumulation, resulting in translocation to the nucleus to increase MITF activity . c-Kit phosphorylation at Y703 and Y936 activates the mitogen-activated protein kinase (MAPK) pathway and leads to cell proliferation, differentiation, and melanin alteration . The activated extracellular signal-related kinase ERK and c-Jun N-terminal kinase JNK, the members of the MAPK family, upregulate the transcription activity or ubiquitin-dependent degradation of MITF as a feedback mechanism of melanin production. On the other hand, the activated p38 induces phosphorylation of CREB and then activates MITF to promote tyrosinase transcription . The other phosphorylation sites such as Y586, Y570, Y900, Y568, Y570, and Y730 are involved in cell proliferation, survival, adhesion, and differentiation, and Y586, Y570, and Y936 participate in receptor downregulation. Some studies even indicated clearly increased expression of SCF was observed after UV light exposure, especially UVB, in both keratinocytes and fibroblasts, which facilitated melanogenesis behavior . In addition, SCF is released in greater levels by fibroblasts than by keratinocytes . Several studies have demonstrated the functions of SCF, irrespective of its secretion by keratinocytes or fibroblasts, in proliferation, differentiation, and melanogenesis of melanocytes; however, the importance of SCF and its effect on other paracrine factors are yet to be determined . Furthermore, fibroblasts play an important role in regulating skin color, and the expression level of SCF in fibroblasts is more than that in keratinocytes. Therefore, it is of particular interest to understand the influence on SCF in terms of whether its dysfunction is occurring in fibroblasts and then the alteration of other cytokines produced by fibroblasts, as well as melanogenesis in melanocytes. This study examined whether SCF silencing influences other paracrine factors secreted by fibroblasts and the changes in melanogenesis of melanocytes.
|
review
| 96.75 |
Transfection was used to introduce siRNA into human fibroblasts. In the attempt to knockdown SCF gene expression, it was necessary to validate how successful its genetic knockdown was. Comparisons between the controlled, regular fibroblast cells, and the same fibroblast cells with introduced 25 nM siRNA delineated the productive knockdown in the mRNA expression levels. Figure 1 shows the qPCR-validated knockdown efficiency of SCF mRNA levels. qPCR is a quantification technique used to determine the mRNA expression levels of, in this case, the amplified SCF gene. Transfection of Hs68 human fibroblasts with 25 nM SCF siRNA showed a significant decrease in SCF gene expression 48 h after the transfection compared with regular Hs68 fibroblasts. Using the transfection method and the qPCR confirmation, the results indicated that a successful >75% of SCF mRNA expression levels were effectively silenced in Hs68 cells.
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study
| 100.0 |
We next examined if SCF inhibited by siRNA caused increased or decreased proliferation of fibroblasts. The MTT method was used to demonstrate fibroblast proliferation. Compared with the vehicle control group, the cell viability showed no difference between the control (100%) and SCF gene knockdown fibroblasts (103%). Figure 1b demonstrates neither cellular morphology nor cell confluency were altered. Similar results were observed regarding fibroblast morphology, where without cellular atrophy damage, flattened or low confluency were observed in Figure 1c. The data indicated the lack of an SCF gene did not affect cell growth and cellular morphology.
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study
| 100.0 |
We evaluated whether SCF affected the fibroblasts and the mRNA paracrine factor levels from fibroblasts. To determine how differently the paracrine factors reacted due to SCF silencing, the controlled gene expressions were compared to the gene expression with SCF knockdown. In Figure 2, the data showed a significant increase in HGF, NRG, and CRH genes when SCF was stably silenced 48 h post transfection. The downregulated mRNA levels with DKK-1 and ET-1 were affected because of SCF knockdown in fibroblast cells. ET-1 was the most severely affected gene, while the paracrine growth factor HGF was the least affected of all the genes. SCF gene alteration affected the fibroblast paracrine system and its knockdown increased or decreased all the specific paracrine gene expression levels.
|
study
| 100.0 |
Paracrine factors showed alterations in their mRNA expression levels when the SCF gene expression was inhibited. Similar experiments under identical conditions were conducted again with the addition of UVB exposure. Exposure to UVB is known to induce expression of SCF, so its effect on mRNA levels compared to the levels without UVB exposure would be a necessary observation. Comparisons between the paracrine factors with and without SCF knockdown, and with or without UVB, emphasize the individual gene expression variations not only in terms of SCF differences but also in determining whether UVB causes certain factors to react and express differently. In Figure 2, the results show the upregulation gene expressions of SCF, HGF, NRG, and CRH with UVB exposure to regular fibroblasts compared with the control group, which did not undergo UVB stimulation. The ET-1 gene was downregulated, and no significant difference was observed with DKK-1. Following the determination of the influence of UVB on regular fibroblasts, the different outcome in fibroblasts with SCF gene knockdown under UVB stimulation was tested. A comparison between regular fibroblasts with UVB treatment and SCF gene knockdown fibroblasts with the same UVB treatment is presented. The data presented the upregulation mRNA expression of HGF, NRG, and CRH and their expression levels when the fibroblast cells, specifically with stable SCF silencing, were exposed to UVB. Compared to the regular fibroblast gene expressions with UVB exposure, HGF, CRH, and NRG with SCF knockdowns all had their mRNA expressions increased, even with the same UVB treated situation. HGF demonstrated a particularly dramatic increase in gene expression levels in cells with SCF genetic knockdown, while DKK-1 and ET-1 were the SCF-silenced genes that were downregulated when treated with UVB. SCF gene alteration influenced the fibroblast paracrine system and the condition differed with or without UVB stimulation.
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study
| 100.0 |
To examine the changes in melanocytes caused by fibroblasts with SCF gene alteration, the genes related to melanogenesis were analyzed. After the fibroblast cells with or without SCF gene were treated with or without UVB, the conditioned medium was collected after 24 h culture and then used to treat melanocytes for 24 h. The experimental conditioned medium obtained from fibroblasts was divided into four experimental groups: fibroblasts without both UVB exposure and SCF inhibition, with only UVB exposure, with only SCF inhibition, and with both UVB exposure and SCF inhibition. These secretions were used as culturing mediums for melanocytes to analyze their different expression levels when cultured in mediums with SCF or without SCF. In Figure 3, SCF gene knockdown, the two conditioned medium groups including the one from regular fibroblasts and SCF-silenced fibroblasts, were used to treat melanocytes separately. The gene expression level of tyrosinase and Pmel17 increased, while MITF, ERK-1, ERK-2, myosin Va (Myo5a), and c-kit decreased. The genes of tyrosinase, ERK-1, ERK-2, Myo5a, and c-kit were upregulated when treated in the conditioned medium with UVB compared with the homeostatic condition. In both treatments of UVB exposure with SCF inhibition group, the data showed the alterations in gene expressions in melanocytes when the conditioned medium was used for treatment. Among them, tyrosinase, MITF, ERK-1, ERK-2, Myo5a, and c-kit were upregulated, and Pmel17 was downregulated when the conditioned medium from fibroblasts undergoing UVB exposure and SCF silencing was applied to treat melanocytes, compared with the same medium with only UVB stimulation. These findings indicated SCF silencing caused the variations in melanocytes and regulated the melanogenesis-related gene expression using the conditioned medium treatment method.
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study
| 100.0 |
After culturing the melanocyte cells in the conditioned mediums containing the fibroblast cell secretions, the foreign medium could potentially have affected melanocytes and melanin production. The alterations in melanin quantities in the melanocytes were confirmed using the melanin content method. Fibroblasts with exposure to UVB, SCF gene knockdown, and SCF gene knockdown with exposure to UVB were compared to figure out the actual variation in melanin content in melanocytes due to the different culturing mediums. In Figure 4a, the melanin production in melanocytes increased by 23.5% when treated in the conditioned medium from SCF fibroblasts knockdown compared to the normal fibroblasts and increased 27.3% under SCF fibroblasts knockdown with UVB exposure. In Figure 4b, the morphology of melanocytes was altered by treatment with the conditioned medium of fibroblasts with SCF knockdown and UVB exposure, resulting in the melanocytes getting more synapses than UVB only. The result was consistent with Figure 2, indicating SCF silencing caused an essential variation in melanin quantities in the melanocytes.
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study
| 100.0 |
Using qPCR, we observed fibroblasts had a c-kit receptor that changed the expression level under different conditions. Figure 5a shows c-kit gene expression was increased with UVB stimulation, even under SCF gene silencing by siRNA transfection compared with regular fibroblasts. In an attempt to demonstrate the existence of an autocrine system in fibroblast cells, different tests were conducted. In Figure 5b, the fibroblast’s inner SCF levels were downregulated when treated with 30 ng of recombinant SCF, while the fibroblast’s inner SCF levels were upregulated when treated with 3 µM of masitinib, which is an inhibitor of SCF and c-kit. When combined, 30 ng of SCF and 3 µM of masitinib produced a result similar to the controlled fibroblast’s inner SCF level. When treated with inhibiting or facilitative SCF environments, fibroblast cells varied their inner SCF levels to balance out their inner and outer levels, confirming the existence of an autocrine signaling system.
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study
| 100.0 |
In human skin, melanosomes are released from melanocyte dendrites and taken directly up by keratinocytes through endocytosis or phagocytosis. Little was known concerning the relationships between the morphology and the melanin content or tyrosinase activities of the melanocytes. In previous keratinocyte and melanocyte co-culture studies, we found that as melanocytes lose their characteristic dendritic structures and adopt fibroblast-like bipolar forms, the cell–cell contact between melanocytes and keratinocytes is considerably reduced, resulting in a reduction in pigment transfer . Our previous two studies suggested that there might be some connections between the dendritic morphology changes and physiological properties of melanocytes . These potential relationships should be further investigated to understand their physiological significances.
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study
| 100.0 |
SCF is a paracrine cytokine that plays an essential role in hematopoiesis, maintenance of cell survival, proliferation, and activation, as well as in melanogenesis. Although several studies demonstrated the functions and mechanisms of SCF in the melanogenesis process, those studies mostly focused on pigment production only. This study examined the role of SCF in paracrine factors in fibroblasts and its subsequent influence on melanocytes under a loss of SCF function in fibroblasts. RNA interference (RNAi) is a physiologically mediated mechanism that induces sequence-specific gene degradation. The double-strand RNA undergoes the dicer processes into the siRNA. Binding of siRNA to the target gene causes mRNA degradation directly or arrests the specific protein translation; therefore, the phenomenon is known as gene silencing . Consequently, siRNA is suitable for observing the gene influences, protein functions, and cell developments when the target gene is knocked down and for understanding the role of the target gene. This study used the SCF-targeted siRNA-cooperated transfection method to introduce siRNA into the intracellular environment and inhibit SCF gene expression in fibroblasts. In this study, all the experiments processed the validation and kept the transfection efficiency at a stable inhibitory state.
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study
| 99.94 |
After siRNA transfected into the intracellular environment and induced cellular SCF gene inhibition, the first aspect to confirm was whether the gene silencing caused cell death or a morphological change, since SCF was responsible for cell survival and proliferation. In MTT assay data, it presented no significant changes in fibroblast cell proliferations, and similar results were observed regarding fibroblast morphology, where without cellular atrophy damage, flattened or low confluency were observed. Fibroblast proliferation did not rely on SCF, even though it was silenced. Although SCF inhibition did not cause fibroblast death, the influence on fibroblasts was unclear. To explore the effect on fibroblasts when the SCF gene was silenced, some paracrine factors secreted by fibroblasts showing effects on melanocyte cell survival and melanin production were determined, including HGF, DKK-1, NRG-1, CRH, and ET-1.
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study
| 100.0 |
HGF is a polypeptide growth factor that can bind to the MET receptor expressed on melanocytes, with HGF stimulation leading to MITF-dependent Met message and protein induction . The MET proto-oncogene encodes for the hepatocyte growth factor (HGF) receptor, a plasma membrane tyrosine kinase that is involved in melanocyte growth and melanoma development. When the ligand binds to the receptor, MET processes autophosphorylation on tyrosine residues produce the docking site for PI3K to promote growth and differentiation of melanocytes, thereby influencing melanogenesis via activation of the cyclic adenosine monophosphate (cAMP) pathway . DKK-1 secreted by fibroblasts mostly exists in the palmoplantar skin compared with the nonpalmoplantar skin . DKK-1 has the ability to inhibit melanocyte growth via regulating the Wnt signaling pathway in which DKK-1 suppresses β-catenin accumulation and then decreases MITF activity, thereby influencing the functions of the melanocytes. In a previous study, Wonseon Choi et al. used the microarray analysis to indicate in dark skin, and NRG-1 was the highly expressed factor secreted by fibroblasts and increased skin pigmentation . NRG-1 mediates melanogenesis through the PI3K pathway, increasing melanocyte proliferation and survival and melanin production. CRH is regulated by UV or as a response to stress. CRH can bind to its receptors, CRH-R1 and CRH-R2, and then induce the POMC-derived hormones, including MSH and ACTH released to influence the skin phenotype system . ET-1 functions as a mitogenic factor for melanocytes and facilitates melanogenesis. ET-1 can bind to its endothelin receptors A (ETA) and activate the cAMP pathway that increases protein kinase C activation to stimulate MITF expression. Akira Hachiya et al. demonstrated the responses of SCF and ET-1 to UVB irradiation, in which SCF responded in the early stage and ET-1 in the later phase of UVB-induced melanin production .
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study
| 99.94 |
Through SCF-targeted siRNA inhibition, we demonstrated HGF, NRG, and CRH gene expressions were significantly increased; especially, HGF gene level had the highest change while DKK-1 and ET-1 gene expressions were decreased. The results demonstrated an autoregulation phenomenon between gene networks, inducing a feedback expression when the SCF gene was silenced in fibroblasts. Cellular autoregulation is frequently observed in gene networks through either negative or positive feedback adjustments . Among them, HGF showed 20-fold high expression in fibroblasts after SCF gene silencing compared with regular fibroblasts. Some experiments demonstrated a similar result that SCF expressed in murine stromal cells was activated and induced an increased expression when treated with extra HGF . This demonstration indicated the relationship between HGF and SCF. Hence, in this case, SCF gene knockdown induced transcription of HGF for facilitating the expression of SCF to balance the gene networks. NRG and CRH involved in melanogenesis also increased their gene expressions under the condition of SCF gene knockdown. On the other hand, DKK-1, a Wnt-antagonist, was downregulated after SCF was inhibited. Gherghe et al. indicated Wnt treatment significantly activated HGF expression in human endothelial progenitor cells . In this case, the Wnt-antagonist DKK-1 was downregulated to avoid the Wnt signaling system inhibition, thereby increasing the expression of HGF. The other downregulated gene, ET-1, was indicated for the mechanism of synergistic effect on ET-1 and SCF that led to melanogenesis. Therefore, SCF inhibition influences ET-1 gene expression.
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study
| 100.0 |
Under the homeostatic status, some paracrine factors are changed or their expression is induced by cells suffering from different stresses such as UV. SCF plays a potential role in regulating melanogenesis under homeostatic and stimulatory statuses, especially in response to UVB irradiation . It is known that UVB upregulates transcription, increases the expressions of c-kit and SCF, and stimulates melanogenic activity in melanocytes. To understand whether the stimulation with UVB when SCF was silenced would influence the paracrine factors in fibroblasts, an experiment with gene knockdown and then treatment with UVB were performed. The results showed fibroblasts under UVB exposure increase the mRNA expression levels of SCF, HGF, DKK-1, NRG, and CRH related to the melanogenesis gene. Under the same condition, ET-1 was downregulated, as shown in previous studies that reported ET-1 was responsible for a later stage of UVB-induced melanin production. Then, comparison between only UVB-treated and both SCF gene knockdown and UVB-stimulated fibroblasts showed similar results but increasing levels of HGF, NRG, and CRH gene expressions under an SCF-silenced status. Among them, HGF showed more than a 3-fold change compared to only UVB-treated fibroblasts and more than a 20-fold change compared to regular fibroblasts. It was demonstrated SCF was important for UVB-induced paracrine secretion, so HGF needed to strengthen its expression to balance the defect from SCF silencing. In addition, SCF and HGF showed a high correlation in fibroblasts with either UVB stimulation or the homeostatic condition .
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study
| 100.0 |
Silencing SCF affected paracrine factor expressions in the fibroblasts. Melanocytes receive the signals generated from fibroblasts and then activate melanin production. In subsequent experiments, we investigated whether SCF-silenced fibroblasts further influenced melanocytes for melanogenesis, and the gene expression in melanocytes was determined. Melanin biosynthesis is a complex process occurring in melanocytes within particular membrane-bound organelles, known as melanosomes . The major component of the melanosome is Pmel17, which is a transmembrane protein and essential for melanin synthesis and deposition to form the fibrillar matrix. Rab27a, melanophilin (MLPH), and Myo5a form a tri-protein complex to bind melanosomes at the melanocytes peripheries. In the process of melanosome transport, the ternary complex is the connection between actin cytoskeleton and melanosome. A lack of these proteins affects the transport, and melanoregulin (Mreg) drives melanosome transfer from melanocytes to keratinocytes via a regulated shedding mechanism. Human skin melanin is driven by the intercellular movement of melanin-containing melanosomes from the extremities of human melanocytes dendrites to neighboring keratinocytes. When it is carried by the actin filament, melanosome moves to the dendritic tail section, through exocytosis, and is transported into keratinocytes . The greater the amount of melanin that is transferred into keratinocytes, the darker is the color of the skin . The movement on the microtubule depends on the dynein–dynactin motor complex. Mreg forms a complex with Rab-interacting lysosomal protein and p150 (Glued), which is a subunit of dynactin . Mreg adjusts a shedding system which makes melanosome transport from human melanocytes to keratinocytes. The shedding process from human melanocytes of melanosome-rich packages is undergoing the phagocytosis of keratinocytes. The shedding not only takes place principally at dendritic extremities, but also around the center areas, having the adhesion to keratinocytes, tightening behind the forming packages, and apparent self-abscissions . The movement on the actin filament requires Myo5a, Rab27a, and MLPH as the connecting bridge . 36H downregulated protein expression for Myo5a and might prevent a darkening of skin color. Melanin is deposited on these fibers and melanosomes become mature in the melanocytes . Melanocytes are activated through multiple paracrine factors secreted from adjacent cells, such as keratinocytes and fibroblasts. The paracrine factors bind to the specific receptors expressed on melanocytes and then accelerate signal transductions to initiate melanin production. For example, SCF binds to the c-kit receptor, and MAPK family, ERK1/2, JNK, and p38, are activated in the melanocytes, thereby altering MITF activities and functions through phosphorylation or dephosphorylation. MITF is a specific transcription factor of tyrosinase, TRP-1 and TRP-2, and the mutant of MITF leads to a deficiency in melanocytes. Melanin is the end product of the multistep conversion of tyrosine. Tyrosinase, a rate-limiting enzyme, catalyzes tyrosine into DOPA, and subsequently DOPA undergoes a separate pathway to convert to pheomelanin or eumelanin. After maturation of melanin, it is transferred to keratinocytes along with microtubules and dendrites by the kinesin and dynein motor proteins. Actin-based motor protein myosinVa attaches to melanosomes and moves via interaction with Rab27a and melanophilin to the surrounding keratinocytes.
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study
| 100.0 |
In this work, to avoid direct UVB exposure and for observing the influence from fibroblasts, culture conditioned mediums from fibroblasts were harvested and used to treat melanocytes. Using the conditioned medium from regular fibroblasts and fibroblasts with UVB treatment, ERK-1, ERK-2, Myo5a, and c-kit genes were upregulated while MITF and Pmel17 were downregulated when treated with UVB-exposed FB medium. According to the above results, UVB-induced SCF, HGF, NRG, and CRH increased. ERK-1 and ERK-2 were significantly increased, attributable to both the SCF and HGF signaling transduction pathways. UVB induced SCF production, so the receptor c-kit expressed on melanocytes increased. In contrast, MITF gene expression was downregulated under UVB-exposed fibroblast medium treatment. McGill et al. indicated MET, the HGF receptor, was regulated by MITF, since MET was a transcriptional target of MITF. In this case, HGF was increased from the conditioned medium and caused high MET expression in the melanocytes, so MITF decreased to reduce MET overexpression .
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study
| 100.0 |
After treatment with the conditioned medium from fibroblasts with SCF silencing, the melanocytes showed downregulation of MITF, ERK-1, ERK-2, Myo5a, and c-kit, while tyrosinase and Pmel17 were upregulated, compared with treatment with regular fibroblast medium. Under this situation, SCF was inhibited by siRNA, so the expression was followed by a decrease, and then the melanocytes treated with this medium showed decreased gene expression of ERK-1, ERK-2, and SCF receptor c-kit. Comparing regular fibroblasts exposed to UVB and SCF-inhibited fibroblasts treated with UVB stimulation, the melanocytes cultured in the conditioned medium showed increased gene expression of tyrosinase, MITF, Myo5a, and c-kit. According to the above results, HGF, NRG, and CRH were upregulated in SCF-silenced fibroblasts with UVB exposure compared with fibroblasts treated with only UVB, so the melanocytes accepted more stimulation to increase the melanogenesis-related gene expression level. SCF silencing also caused ERK-1 downregulation, but ERK-2 showed no significant change in its gene level compared to regular fibroblasts exposed to UVB in melanocytes. In addition, this result is observed when the SCF gene is knocked down, the gene Myo5a is downregulated, and Pmel17 is upregulated whether there is UVB exposure or not.
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study
| 100.0 |
To confirm the essential changes in the melanocyte, the quantity of melanin production was examined. The melanin content assay showed melanocytes cultured with conditioned medium from SCF-silenced fibroblasts exposed to UVB increased the amount of melanin by about 18%, compared with regular fibroblasts exposed to UVB. This result was consistent with the abovementioned data that melanogenesis-related genes were upregulated. Based on these findings, indeed, SCF silencing caused variations in fibroblasts to paracrine factors, and the variation changed if fibroblasts suffered UVB stimulation. The results from different fibroblasts affected melanocyte activities via melanogenesis-related gene expression. Especially under the condition of fibroblasts with SCF silencing and then exposure to UVB, the melanocytes cultured in fibroblast secretions exhibited increased melanogenesis-related gene levels, and the phenomenon was confirmed by their increased melanin quantities . Analysis of the SCF receptor c-kit mRNA expression level in the fibroblasts showed the c-kit gene level increased under SCF inhibition in fibroblasts and further increased when fibroblasts with SCF inhibition were exposed to UVB. Although c-kit showed increased expression following UVB exposure with or without SCF gene knockdown, the underlying mechanisms need to be confirmed. A probable reason would be that as SCF and c-kit were induced by UVB stimulation and SCF was knocked down, c-kit increased its expression to improve the binding of the ligand to the receptor.
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study
| 100.0 |
Autocrine signaling is a type of cell regulation in which cells secrete and respond to their particular growth factors. According to fibroblast autoregulated gene expression under SCF gene knockdown and c-kit expression on fibroblasts, the fibroblasts regulated SCF produced in an autocrine manner. Addition of recombinant SCF resulted in a decreased expression level, showing a natural feedback in that the fibroblast cells stopped producing as much SCF . Similarly, adding masitinib inhibited the binding between SCF and c-kit, and so the expression level was increased. The fact that adding both masitinib and SCF produces similar expression levels as the control indicated the automatic balancing of the inner SCF levels, which is the role of an autocrine signaling system. A similar SCF autocrine effect was demonstrated in several tumors . These results further showed the existence of an autocrine system with co-expression of SCF and c-kit in fibroblasts. The present study revealed that although SCF is involved in several processes such as cellular differentiation, proliferation, and survival, it does not influence fibroblast proliferation. Once SCF is silenced in fibroblasts, the autoregulation in gene networks balances the SCF defects in an autocrine regulatory manner. Investigation of the paracrine factors HGF, DKK-1, NRG, CRH, and ET-1 showed HGF exhibited an especially high correlation with SCF. Fibroblasts with SCF dysfunction influenced their own paracrine systems and further affected melanocytes to alter melanin production, particularly under UVB stimulation (Figure 6). Altogether, SCF plays an essential role in fibroblast paracrine factors and in the melanocyte cellular melanogenesis effect.
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study
| 100.0 |
Cell culture materials Medium 254, Medium 254 supplement, fetal bovine serum (FBS), Dulbecco’s modified Eagle medium (DMEM), and antibiotics were obtained from Gibco (Waltham, MA, USA). Short interfering RNA (siRNA) transfection reagent, Trizol reagent, 1-bromo-3-chloropropane (BCP), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical (St. Louis, MO, USA). ToolScript MMLV RT kit and TOOLS 2× SYBR quantitative real-time polymerase chain reaction (qPCR) Mix was purchased from Biotools (Taipei, Taiwan).
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other
| 99.9 |
The neonatal foreskin human melanocytes purchased from Cascade Biologics (C-102-5C, Gibco) were maintained in Medium 254 supplemented with human melanocyte growth supplement containing basic FGF (3 ng/mL), insulin (5 µg/mL), transferrin (5 µg/mL), bovine pituitary extract (0.2%), FBS (0.5%), heparin (3 µg/mL), hydrocortisone (0.18 µg/mL), and phorbol 12-myristate 13-acetate (10 ng/mL). The human fibroblasts Hs68 cells derived from male foreskin (ATCC® CRL-1635™) were obtained from the Food Industry Research and Development Institute (FIRDI, Hsinchu, Taiwan) and cultured in DMEM supplemented with 10% FBS and 1% antibiotic. All cells were incubated at 37 °C under 5% CO2 .
|
study
| 99.9 |
Fibroblasts were seeded in a 6-well plate at a density of 2 × 105 cells and then cultured for 24 h until the cells reached about 80%–90% confluence. Then, the siRNA mixture was prepared for further experiments. SCF-targeted pooled siRNA supplied by Dharmacon (GE Dharmacon, Lafayette, CO, USA) was diluted to 25 nM in 100 µL per well of serum-free DMEM and 20 µL per well transfection reagent was added into the diluted siRNA solution. After mixing and incubation for 20 min, 100 µL of the mixture was added to each well and incubated for 3 days at 37 °C under 5% CO2. The control group was fibroblasts transfected reagent without siRNA. The transgene expression efficiency was detected by qPCR. The siRNA sequences used in the experiment are shown in Table 1.
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study
| 100.0 |
MTT assay was used to evaluate cell viability and proliferation. MTT, a tetrazole with a yellow color, is taken up by living cells and then converted into a purple formazan-reduced state by mitochondrial dehydrogenase. Cells were seeded in a 96-well plate at a density of 8 × 103 cells per well and cultured for 24 h. Following attachment, cells were replaced in fresh medium with siRNA and transfection reagent complex and cultured for 48 h. Then, the medium was changed to fresh medium containing 100 µL of 0.5 mg/mL MTT and then cultured for 2 h at 37 °C under 5% CO2. Then, the medium was discarded and 100 µL DMSO was added to dissolve the formazan. The absorbance was measured at 595 nm .
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study
| 100.0 |
To induce SCF secretion from fibroblasts and simulate the condition of light stimulation, UVB was used to complete the test. This test was performed according to the method, with some modifications, described by Shin et al. . Fibroblasts with or without previous siRNA transfection were cultured in a 6-well plate. Before UV exposure, the medium was replaced by PBS during that time. UVB exposure was performed using Ultraviolet Crosslinkers CL-1000 (UVP, Upland, CA, USA) with UVB radiation (302 nm) at a nontoxic dosage of 5 mJ/cm2 3 times at intervals of 5 h. After UVB treatment and culturing for 24 h, the medium was collected into the conditioned medium and used for treating melanocytes previously cultured in the 6-well plate for 24 h. Experimental cells were harvested for subsequent testing.
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study
| 100.0 |
The total RNA was extracted using Trizol RNA isolation reagent, which can break down cell lysates and isolate RNA, DNA, and proteins. First, 1 mL Trizol reagent was added to each and transferred to a 1.5-mL microtube at room temperature for 5 min. Then, 200 µL BCP per mL of Trizol reagent was added and mixed vigorously. Following incubation for 2 min, the samples were centrifuged at 14,000× g for 15 min. The sample homogenates formed two phases, from which the aqueous phase on the top of the homogenate was transferred to a new Eppendorf tube. To precipitate RNA, an equal volume of isopropanol was added and mixed. The mixture was centrifuged at 14,000× g for 15 min and the supernatant was removed. The RNA pellet was washed with 1 mL of 75% ethanol to remove the residual salts. Finally, the mixture was centrifuged at 12,000× g for 5 min and the RNA pellet was dried and dissolved with 50 µL diethylpyrocarbonate (DEPC)-treated water. The concentration and quality of the RNA extracts were determined by NanoDrop (Thermo Fisher, Waltham, MA, USA).
|
study
| 99.94 |
Before qPCR analysis, RNA should be processed by a reverse transcription step to obtain complementary DNA (cDNA). cDNA synthesis was accomplished by ToolScript MMLV RT kit. Briefly, 1 µg total RNA of each sample was mixed with 2 µL oligo dT, 2 µL dNTP, and distillation-distillation H2O to bring the total volume to 14.5 µL. To open the RNA secondary structure, the RNA mixture was heated at 70 °C for 5 min and then cooled on ice for 2 min. To the cooled mixture were added 4 µL reverse buffer, 0.5 µL RNasin, and 1 µL MMLV. Afterward, the mixture was incubated at 42 °C for 50 min to synthesize cDNA and then heated at 95 °C for 5 min to inactivate the MMLV. Further, the cDNA products were processed on qPCR using TOOLS 2× SYBR qPCR Mix. First, a reaction solution was prepared containing 10 µL 2× SYBR qPCR mix, 0.1 µL forward primer, 0.1 µL reverse primer, 1 µL cDNA template, 2 µL of 50× ROX reference dye, and distillation–distillation H2O to bring the total volume to 20 µL per well. Next, the qPCR machine (ABITM StepOneTM Plus, Thermo Fisher) was set up. The initial denaturation temperature was 95 °C for 15 min by one cycle, followed by a denaturation temperature of 95 °C for 10 s, annealing at 60 °C for 20 s, and extension at 72 °C for 30 s, with the total PCR stage being 40 cycles. The designed forward and reverse primers from 5′ to 3′ used in this experiment are shown in (Table 2) .
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study
| 99.94 |
Estimation of melanin content is a method for cellular melanin determination. At first, cells were seeded in a 6-well plate at a density of 2 × 105 cells and cultured for 24 h. Then, the medium was changed to conditioned medium obtained from fibroblasts cultured for 24 h. Afterward, the cell pellets were harvested and dissolved in 2.0 N NaOH, and then heated at 95 °C for 1 h. The absorbance of melanin was measured at 490 nm .
|
study
| 99.94 |
All the experiments in each platform were carried out in triplicate and presented as mean ± standard error. For statistical analysis, all data were analyzed by Student’s t-test for multiple comparisons. A significant difference (*) was defined as p < 0.05.
|
study
| 99.94 |
Systemic lupus erythematosus (SLE) is one of the representative systemic autoimmune diseases, which is characterized by the presence of autoantibodies and involves virtually any organ. Lupus nephritis represents a crucial complication because renal outcomes in patients with lupus nephritis are poor despite the use of immunosuppressive therapy1. A previous study has shown that approximately one-third of patients with lupus nephritis progress to end-stage renal disease within 20 years2. An understanding of its detailed pathogenesis is urgently needed to find better therapeutic approaches.
|
review
| 99.8 |
Mouse natural killer T (NKT) cells are innate immune cells that express both the NK1.1 antigen and the T cell receptor (TCR) and are abundant in the liver. Once activated by IL-12 or an anti-CD3 antibody (Ab), they produce cytokines and exert antitumor cytotoxicity3,4. Their TCRs are mainly encoded by the Vα14Jα18 and Vβ8 genes5,6. The gene arrangement for encoding the TCR is invariant, and therefore, they are also known as invariant NKT (iNKT) cells. The functions of NKT cells have been examined under stimulation with alpha-galactosylceramide (α-GalCer), a specific sphingoglycolipid ligand of these cells7.
|
study
| 100.0 |
Liver NKT cells activated by α-GalCer trigger a potent antitumor response mediated via NK cells and subsequently CD8+ T cells8,9. However, NKT cells themselves can cause multiple organ failure, especially in aged mice8,10. On the other hand, NKT cells may play protective roles in some glomerulonephritis or vasculitis models11,12. In a SLE model, the expansion of NKT cells is thought to be involved in the onset of lupus nephritis13, whereas their immunoregulatory roles were also reported both in human SLE and SLE models14–16.
|
review
| 99.6 |
The effects of α-GalCer in the progression of autoimmune disease models have also been controversial. For example, it was shown that α-GalCer prevented the onset of diabetes in non-obese diabetic (NOD) mice, a representative type 1 diabetes model17,18. Conversely, there have been contradictory reports regarding the effects of α-GalCer in NZB/NZW F1 (BWF1) mice, an experimental model of SLE in which lupus nephritis-like lesions develop. Thus, one report showed that α-GalCer-activated NKT cells exacerbated the experimental lupus nephritis19, whereas a long-term reduction in severe proteinuria following α-GalCer treatment was reported in another study20.
|
review
| 99.8 |
Therefore, in the present study, we have investigated the roles of NKT cells in lupus nephritis using BWF1 mice. We show that the repeated administration of α-GalCer into BWF1 mice induced beneficial effects, as follows: (i) it improved proteinuria which represents a hallmark of renal injury, by protecting nephrin, a key functional molecule in the slit diaphragm of the podocytes21; (ii) it suppressed B cell function and decreased glomerular immune complex deposits; (iii) it induced not only an anergic state to α-GalCer in NKT cells, but also decreased the number of NKT cells in multiple organs and the production of IL-4 by these cells.
|
study
| 100.0 |
The levels of proteinuria in the α-GalCer group were significantly lower than those in the vehicle group (Fig. 1a). Moreover, the incidence of proteinuria in the α-GalCer group was also significantly lower when analyzed using the log-rank test (Fig. 1b, p < 0.05), but was not statistically significant when using the Cox proportional hazard model (Supplementary Table S1, p = 0.10). Neither group displayed hematuria. The survival rate in the α-GalCer group tended to be higher than that in the vehicle group, although it was not statistically different (Fig. 1c, p = 0.20 and Supplementary Table S1, p = 0.33).Figure 1The effects of repeated alpha-galactosylceramide (α-GalCer) administration on proteinuria and survival rates. (a) The levels of proteinuria in each group, measured 8 weeks after the last α-GalCer or vehicle injection (n = 5–7 in each group). *p < 0.05 compared with the vehicle group. (b) The percentages of mice with free of proteinuria and (c) the survival rate in the α-GalCer and the vehicle groups (n = 8 and 10, respectively). Arrows show the timing of when the mice were injected with α-GalCer or vehicle.
|
study
| 100.0 |
The effects of repeated alpha-galactosylceramide (α-GalCer) administration on proteinuria and survival rates. (a) The levels of proteinuria in each group, measured 8 weeks after the last α-GalCer or vehicle injection (n = 5–7 in each group). *p < 0.05 compared with the vehicle group. (b) The percentages of mice with free of proteinuria and (c) the survival rate in the α-GalCer and the vehicle groups (n = 8 and 10, respectively). Arrows show the timing of when the mice were injected with α-GalCer or vehicle.
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study
| 100.0 |
The blood urea nitrogen (BUN) levels in the α-GalCer group were significantly lower than those in the vehicle group (Table 1). Moreover, the serum albumin levels in the α-GalCer group were significantly higher than those in the vehicle group, reflecting the difference in the levels of proteinuria between the two groups (Fig. 1a). Although no significant differences in the total serum protein levels or IgM levels were observed, the serum albumin/globulin ratio was significantly higher in the α-GalCer group. However, the serum IgG anti-dsDNA Ab levels did not differ between the two groups. The alanine aminotransferase levels were within the normal range and no significant difference was found between the two groups. Splenomegaly was observed in both groups; however, there was no difference in spleen weight or body weight. Furthermore, obvious lymphadenopathy was not observed in any of the mice.Table 1Biochemical and physiological parameters at the end of the experiment.Vehicle groupα-GalCer groupNumber57Blood urea nitrogen (mg/dL)41.0 ± 34.017.3 ± 5.6*Serum albumin (g/dL)1.84 ± 0.312.24 ± 0.21*Total protein (g/dL)5.86 ± 0.216.09 ± 0.47IgM (mg/dL)269 ± 44200 ± 16Albumin/globulin ratio0.46 ± 0.110.58 ± 0.03*Serum IgG anti-dsDNA Ab (U/mL)1734 ± 18041805 ± 1256Alanine aminotransferase (IU/L)28.0 ± 3.624.6 ± 3.6Spleen weight (g)0.18 ± 0.050.22 ± 0.04Body weight (g)39.8 ± 1.038.9 ± 1.3Data are presented as mean ± SEM or number.*p < 0.05 (Student’s t-test).α-GalCer: alpha-galactosylceramide.
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| 100.0 |
A representative image of periodic acid-Schiff (PAS) staining in the vehicle group is shown in Fig. 2a, while that in the α-GalCer group is shown in Fig. 2b. The percentage of glomeruli containing immune complex deposits and that of sclerotic glomeruli in the α-GalCer group were significantly lower (Fig. 2c,d). Furthermore, although the difference was not statistically significant, the percentage of glomeruli with crescents in the α-GalCer group tended to be lower than that in the vehicle group (Fig. 2e).Figure 2Kidney pathology of experimental lupus nephritis is ameliorated by treatment with alpha-galactosylceramide (α-GalCer). Representative images of periodic acid-Schiff staining of kidney slices in the (a) vehicle and (b) α-GalCer groups. Arrows, immune complex deposits; arrowhead, segmentally sclerotic area. The percentages of (c) glomeruli with immune complex deposits, (d) sclerotic glomeruli, and (e) glomeruli with crescents in each group (vehicle group; n = 5, α-GalCer group; n = 7). *p < 0.05 compared with the vehicle group.
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study
| 100.0 |
Kidney pathology of experimental lupus nephritis is ameliorated by treatment with alpha-galactosylceramide (α-GalCer). Representative images of periodic acid-Schiff staining of kidney slices in the (a) vehicle and (b) α-GalCer groups. Arrows, immune complex deposits; arrowhead, segmentally sclerotic area. The percentages of (c) glomeruli with immune complex deposits, (d) sclerotic glomeruli, and (e) glomeruli with crescents in each group (vehicle group; n = 5, α-GalCer group; n = 7). *p < 0.05 compared with the vehicle group.
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study
| 100.0 |
We also evaluated the infiltration of inflammatory cells in the kidney. Representative images for F4/80 and CD3 staining in both groups are shown in Fig. 3a,b. Both the F4/80 and CD3 positive areas were significantly smaller in the α-GalCer group than those in the vehicle group (Fig. 3c,d). The renal messenger RNA (mRNA) expression levels for a dendritic cell marker (CD11c) and a plasma cell marker (CD138) did not significantly differ between the two groups (Supplementary Fig. S1).Figure 3Alpha-galactosylceramide (α-GalCer) treatment decreases the infiltration of inflammatory cells into the kidney. Representative images of (a) F4/80-stained and (b) CD3-stained kidney slices in the vehicle (left) and α-GalCer (right) groups. In each panel, arrows indicate F4/80 or CD3-positive areas. The percentages of the (c) F4/80-positive area and (d) CD3-positive area in each group (vehicle group; n = 5, α-GalCer group; n = 7). *p < 0.05 compared with the vehicle group.
|
study
| 100.0 |
Alpha-galactosylceramide (α-GalCer) treatment decreases the infiltration of inflammatory cells into the kidney. Representative images of (a) F4/80-stained and (b) CD3-stained kidney slices in the vehicle (left) and α-GalCer (right) groups. In each panel, arrows indicate F4/80 or CD3-positive areas. The percentages of the (c) F4/80-positive area and (d) CD3-positive area in each group (vehicle group; n = 5, α-GalCer group; n = 7). *p < 0.05 compared with the vehicle group.
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study
| 100.0 |
Next, we assessed the deposition of glomerular immune complexes and the podocyte damage of the two groups. Figure 4a shows representative images of immunofluorescence (IF) IgG staining. In accordance with the light microscopy findings, the mean fluorescence intensity (MFI) of IgG staining in the α-GalCer group was significantly weaker (Fig. 4b).Figure 4Alpha-galactosylceramide (α-GalCer) treatment decreases the deposition of glomerular immune complexes and ameliorates podocyte injury. Representative images of (a) immunofluorescence (IF) IgG staining, (c) immunoperoxidase WT-1 staining, and (e) IF nephrin staining of kidney slices in the vehicle (left) and α-GalCer (right) groups. In (c) and (e), arrows indicate positively stained cells or areas. (b) MFI (mean fluorescence intensity) of IgG staining, (d) WT-1 positive cells/glomeruli, and (f) semiquantitative nephrin IF score in each group (vehicle group; n = 5, α-GalCer group; n = 7). *p < 0.05, **p < 0.01 compared with the vehicle group. (g) Representative electron microscopy photomicrographs of kidney slices in the vehicle (left) and α-GalCer (right) groups. Widespread effacement of podocyte foot processes (red arrows) and electron dense immune deposits (red arrowheads) are shown in the vehicle group. Endocapillary infiltration of inflammatory cells was also observed (yellow asterisks).
|
study
| 100.0 |
Alpha-galactosylceramide (α-GalCer) treatment decreases the deposition of glomerular immune complexes and ameliorates podocyte injury. Representative images of (a) immunofluorescence (IF) IgG staining, (c) immunoperoxidase WT-1 staining, and (e) IF nephrin staining of kidney slices in the vehicle (left) and α-GalCer (right) groups. In (c) and (e), arrows indicate positively stained cells or areas. (b) MFI (mean fluorescence intensity) of IgG staining, (d) WT-1 positive cells/glomeruli, and (f) semiquantitative nephrin IF score in each group (vehicle group; n = 5, α-GalCer group; n = 7). *p < 0.05, **p < 0.01 compared with the vehicle group. (g) Representative electron microscopy photomicrographs of kidney slices in the vehicle (left) and α-GalCer (right) groups. Widespread effacement of podocyte foot processes (red arrows) and electron dense immune deposits (red arrowheads) are shown in the vehicle group. Endocapillary infiltration of inflammatory cells was also observed (yellow asterisks).
|
study
| 100.0 |
Figure 4c shows representative images of WT-1 immunostaining, which represents a marker of podocyte damage22. Compared with that in the vehicle group, the number of WT-1 positive cells was significantly larger in the α-GalCer group (Fig. 4d). The IF nephrin staining score was also significantly higher in the α-GalCer group (Fig. 4e,f). These results suggested that podocyte injury was alleviated in the α-GalCer group.
|
study
| 100.0 |
Moreover, electron microscopy showed that although widespread effacement of podocyte foot processes was observed in the vehicle group, foot processes were relatively preserved in the α-GalCer group (Fig. 4g). Electron dense deposits, especially subepithelial deposits, were also smaller in the α-GalCer group.
|
study
| 100.0 |
We performed flow cytometry analysis to assess the number of NKT cells in multiple organs. Figure 5a shows representative images in which liver mononuclear cells (MNCs) were stained with anti-TCR αβ and anti-NK1.1 Abs. The proportion of NKT cells in the liver in the α-GalCer group was significantly reduced compared to that in the vehicle group (Fig. 5b). Figure 5c,e and g show representative images in which liver, kidney, and spleen MNCs were stained with anti-TCR αβ Ab and the α-GalCer-loaded CD1d tetramer. The proportions of iNKT cells in the liver, kidney, and spleen in the α-GalCer group were significantly lower than those in the vehicle group (Fig. 5d,f and h).Figure 5Repeated alpha-galactosylceramide (α-GalCer) administration decreases the proportion of NKT cells in multiple organs. (a) Flow cytometry analysis of liver mononuclear cells (MNCs) in the vehicle (left) and α-GalCer (right) groups, stained with anti-TCR αβ and anti-NK1.1 antibodies. In both panels, black squares show NKT cells. Flow cytometry analysis of (c) liver, (e) kidney, and (g) spleen MNCs in the (left) vehicle and (right) α-GalCer groups. Isolated MNCs were double stained with anti-TCR αβ antibody and α-GalCer-loaded CD1d tetramer; invariant NKT (iNKT) cells are shown in black squares. The percentages of (b) whole NKT cells in the liver and iNKT cells in the (d) liver, (f) kidney, and (h) spleen are shown (n = 4 in each group). *p < 0.05 compared with the vehicle group.
|
study
| 100.0 |
Repeated alpha-galactosylceramide (α-GalCer) administration decreases the proportion of NKT cells in multiple organs. (a) Flow cytometry analysis of liver mononuclear cells (MNCs) in the vehicle (left) and α-GalCer (right) groups, stained with anti-TCR αβ and anti-NK1.1 antibodies. In both panels, black squares show NKT cells. Flow cytometry analysis of (c) liver, (e) kidney, and (g) spleen MNCs in the (left) vehicle and (right) α-GalCer groups. Isolated MNCs were double stained with anti-TCR αβ antibody and α-GalCer-loaded CD1d tetramer; invariant NKT (iNKT) cells are shown in black squares. The percentages of (b) whole NKT cells in the liver and iNKT cells in the (d) liver, (f) kidney, and (h) spleen are shown (n = 4 in each group). *p < 0.05 compared with the vehicle group.
|
study
| 100.0 |
Next, we assessed the cytokine responses under α-GalCer stimulation. The repeated administration of α-GalCer reduced the elevation of serum IFN-γ and IL-4 levels following α-GalCer injection in mice, and the levels of both cytokines decreased to undetectable levels (Fig. 6a,b). These results were also confirmed in vitro. Thus, α-GalCer-stimulated liver MNCs strongly proliferated in the vehicle group (Fig. 6c), whereas the proliferation was poor in the α-GalCer group (Fig. 6d). In addition, the IFN-γ and IL-4 levels produced by liver MNCs following α-GalCer stimulation were significantly lower in the α-GalCer group, even at 8 weeks after the last α-GalCer injection (Fig. 6e,f). Furthermore, not only BWF1, but also C57BL/6 J (B6) mice undergoing repeated α-GalCer administration showed similar cytokine responses both in in vivo and in vitro (Fig. 6a,b,e and f). These results suggested that the repeated administration of α-GalCer induced a state of anergy to α-GalCer, which was not specific to BWF1 mice.Figure 6Repeated alpha-galactosylceramide (α-GalCer) injection induces anergy in NKT cells to α-GalCer. (a) Serum IFN-γ levels 12 hours after α-GalCer injection and (b) IL-4 levels 3 hours after α-GalCer injection in (NZB/NZW) F1 (BWF1) and C57BL/6 J (B6) mice. The mice were injected with 2 μg/body of α-GalCer once a week for 4 weeks, and cytokine levels were evaluated after every injection (n = 4 in each group). *p < 0.05, **p < 0.01 compared with B6 mice. ††p < 0.01 compared with the levels after the first α-GalCer injection. †p < 0.05 compared with the levels after the second α-GalCer injection. Microphotographs of liver mononuclear cells (MNCs), stimulated by α-GalCer in the (c) vehicle and (d) α-GalCer groups (original magnification, 100×). In vitro production of (e) IFN-γ and (f) IL-4 from liver MNCs in BWF1 mice treated with α-GalCer or vehicle, and B6 mice treated with α-GalCer. Liver MNCs were obtained 8 weeks after the last α-GalCer or vehicle injection and were stimulated with α-GalCer for 48 hours (n = 4 in each group). **p < 0.01 compared with the vehicle group of BWF1 mice.
|
study
| 100.0 |
Repeated alpha-galactosylceramide (α-GalCer) injection induces anergy in NKT cells to α-GalCer. (a) Serum IFN-γ levels 12 hours after α-GalCer injection and (b) IL-4 levels 3 hours after α-GalCer injection in (NZB/NZW) F1 (BWF1) and C57BL/6 J (B6) mice. The mice were injected with 2 μg/body of α-GalCer once a week for 4 weeks, and cytokine levels were evaluated after every injection (n = 4 in each group). *p < 0.05, **p < 0.01 compared with B6 mice. ††p < 0.01 compared with the levels after the first α-GalCer injection. †p < 0.05 compared with the levels after the second α-GalCer injection. Microphotographs of liver mononuclear cells (MNCs), stimulated by α-GalCer in the (c) vehicle and (d) α-GalCer groups (original magnification, 100×). In vitro production of (e) IFN-γ and (f) IL-4 from liver MNCs in BWF1 mice treated with α-GalCer or vehicle, and B6 mice treated with α-GalCer. Liver MNCs were obtained 8 weeks after the last α-GalCer or vehicle injection and were stimulated with α-GalCer for 48 hours (n = 4 in each group). **p < 0.01 compared with the vehicle group of BWF1 mice.
|
study
| 100.0 |
The cytokine production levels of MNCs were also evaluated under stimulation with other agents. Liver MNCs stimulated with concanavalin A (ConA), a T-cell stimulant23, proliferated well both in the vehicle and α-GalCer groups (Fig. 7a,b), and the levels of IFN-γ produced by these cells were comparable between the two groups (Fig. 7c). However, the IL-4 levels in the α-GalCer group were significantly lower than those in the vehicle group (Fig. 7d). Similar cytokine responses were observed when liver MNCs were stimulated with an anti-CD3 Ab. Although the levels of IFN-γ produced by liver MNCs did not differ between the two groups, the IL-4 levels in the α-GalCer group were significantly lower than those in the vehicle group (Fig. 7e,f). This suppression of IL-4 production was also observed in B6 mice, following the repeated administration of α-GalCer (Fig. 7c–f).Figure 7Th2 but not Th1 immune responses are suppressed by repeated alpha-galactosylceramide (α-GalCer) injections. Microphotographs of liver mononuclear cells (MNCs), stimulated by concanavalin A (ConA) in the (a) vehicle and (b) α-GalCer groups (original magnification, 100×). IFN-γ production and IL-4 production from liver MNCs, stimulated with (c,d) ConA or (e,f) anti-CD3 antibody for 48 hours in (NZB/NZW) F1 (BWF1) mice treated with α-GalCer or vehicle, and C57BL/6 J (B6) mice treated with α-GalCer (n = 4–5 in each group). **p < 0.01, ***p < 0.001 compared with the vehicle group of BWF1 mice. ††p < 0.01 compared with the α-GalCer group of BWF1 mice.
|
study
| 100.0 |
Th2 but not Th1 immune responses are suppressed by repeated alpha-galactosylceramide (α-GalCer) injections. Microphotographs of liver mononuclear cells (MNCs), stimulated by concanavalin A (ConA) in the (a) vehicle and (b) α-GalCer groups (original magnification, 100×). IFN-γ production and IL-4 production from liver MNCs, stimulated with (c,d) ConA or (e,f) anti-CD3 antibody for 48 hours in (NZB/NZW) F1 (BWF1) mice treated with α-GalCer or vehicle, and C57BL/6 J (B6) mice treated with α-GalCer (n = 4–5 in each group). **p < 0.01, ***p < 0.001 compared with the vehicle group of BWF1 mice. ††p < 0.01 compared with the α-GalCer group of BWF1 mice.
|
study
| 100.0 |
To assess the B cell function, we measured the IgM production levels of splenic MNCs. The number of B cells was also evaluated by flow cytometry. In the α-GalCer group, the expression of Toll-like receptor-9 (TLR-9) which recognizes the CpG-oligodeoxynucleotide (CpG-ODN), common bacterial DNA, in splenic F4/80+ CD11b+ macrophages was significantly lower (Fig. 8a). Moreover, the IgM levels produced by spleen MNCs following CpG-ODN stimulation were significantly lower (Fig. 8b), and those produced by lipopolysaccharide (LPS)-stimulated spleen MNCs also tended to be lower (Fig. 8c). The proportions of B cells in the liver, kidney, and spleen did not differ between the two groups (Supplementary Fig. S2).Figure 8A decrease in B cell function and augmentation of NK cell function is observed in (NZB/NZW) F1 (BWF1) mice treated with alpha-galactosylceramide (α-GalCer). (a) Representative histograms obtained by flow cytometry showing the expression of intracellular Toll-like receptor-9 (TLR-9) in splenic F4/80+ CD11b+ macrophages in the vehicle (left) and α-GalCer (right) groups of BWF1 mice. The data represent the mean ± SEM in each group (n = 4 in each group). IgM production by spleen mononuclear cells (MNCs) stimulated with (b) CpG-oligodeoxynucleotide (CpG-ODN) or (c) lipopolysaccharide for 48 hours in each group (n = 4–6 in each group). (d) IFN-γ production by liver MNCs stimulated by CpG-ODN for 48 hours in each group (n = 4 in each group). *p < 0.05 compared with the vehicle group.
|
study
| 100.0 |
A decrease in B cell function and augmentation of NK cell function is observed in (NZB/NZW) F1 (BWF1) mice treated with alpha-galactosylceramide (α-GalCer). (a) Representative histograms obtained by flow cytometry showing the expression of intracellular Toll-like receptor-9 (TLR-9) in splenic F4/80+ CD11b+ macrophages in the vehicle (left) and α-GalCer (right) groups of BWF1 mice. The data represent the mean ± SEM in each group (n = 4 in each group). IgM production by spleen mononuclear cells (MNCs) stimulated with (b) CpG-oligodeoxynucleotide (CpG-ODN) or (c) lipopolysaccharide for 48 hours in each group (n = 4–6 in each group). (d) IFN-γ production by liver MNCs stimulated by CpG-ODN for 48 hours in each group (n = 4 in each group). *p < 0.05 compared with the vehicle group.
|
study
| 100.0 |
On the other hand, the IFN-γ levels produced by liver MNCs following stimulation with CpG-ODN were significantly higher in the α-GalCer group (Fig. 8d), suggesting that the function of NK cells was enhanced24. TLR-9 expression in hepatic F4/80+ CD11b+ macrophages did not differ between the two groups (data not shown).
|
study
| 100.0 |
To our knowledge, this is the first report to demonstrate that a repeated α-GalCer injection ameliorates podocyte injury, thereby inducing an anti-proteinuric effect. The number of WT-1 positive cells was significantly larger and the nephrin staining score was significantly higher in the α-GalCer group. Moreover, the preservation of podocyte foot processes was observed in the α-GalCer group. We assessed the proteinuria-free survival time in mice using two analysis methods; one showed a statistically significant difference, while another only suggested a tendency toward significance. Regarding this point, it has been reported that the log-rank test may show a tendency toward significant results25. Regardless, the daily levels of proteinuria in the α-GalCer group were significantly lower than those in the vehicle group.
|
study
| 100.0 |
In our study, the infiltration of F4/80-positive macrophages and CD3-positive T lymphocytes in the kidney was reduced, and the serum albumin/globulin ratio was higher in the α-GalCer group. A low albumin/globulin ratio reflects chronic inflammation26. Therefore, our data also suggested the resolution of inflammation following the repeated administration of α-GalCer.
|
study
| 100.0 |
It has been reported that NKT cells and B cells interact to generate Igs, including autoantibodies27,28, whereas inhibitory roles of NKT cells against autoreactive B cells have also been described29. In the present study, we showed another novel finding that the repeated administration of α-GalCer decreased glomerular immune complex deposits. Thus, the percentage of glomeruli containing immune complex deposits, as assessed by PAS staining, was significantly lower and the IgG deposition was significantly decreased in the α-GalCer group. These findings were also observed by electron microscopy. Moreover, the IgM levels produced by CpG-ODN-stimulated spleen MNCs were significantly lower. These data suggested the suppression of autoimmune responses mediated by a decrease in B cell function. The decreased TLR-9 expression of splenic macrophages in the α-GalCer group may also have been related to the improvement of kidney pathology, since TLR-9 has been implicated in the development of lupus nephritis30. Despite the alleviation of renal injuries, the administration of α-GalCer did not affect the levels of anti-dsDNA IgG Abs. However, in the present study, the levels of anti-dsDNA Abs did not correlate with the levels of proteinuria in BWF1 mice, suggesting that these autoantibodies are not pathogenetic markers. Similarly, a study using another SLE model demonstrated an improvement in skin lesions, without any changes in the levels of anti-dsDNA Abs31.
|
study
| 99.94 |
As previously reported32, a repeated α-GalCer injection induced anergy in NKT cells. In addition, the proportions of NKT cells in the liver, kidney, and spleen were significantly reduced in the α-GalCer group. In contrast, the IFN-γ levels remained constant following stimulation with ConA and anti-CD3 Abs, showing that the anergy observed in NKT cells was specific to α-GalCer.
|
study
| 100.0 |
On the other hand, the IL-4 levels produced by ConA and anti-CD3-stimulated liver MNCs were significantly reduced in the α-GalCer group, suggesting the suppression of Th2 immune responses. A predominant Th2 cytokine response in human membranous lupus nephritis has been reported33, and important roles for the Th2 immune responses in the pathogenesis of membranous lupus nephritis have been suggested34,35. Therefore, the present study strongly supported that the repeated administration of α-GalCer ameliorated the progression of lupus nephritis through the suppression of Th2 immune responses. However, it has also been reported that an α-GalCer injection modulates immune responses towards a Th2 phenotype in B6 mice36 and that Th2-biased immune responses induced by the administration of α-GalCer17,18 or its derivative37 prevented diabetes in NOD mice. Thus, the effects of α-GalCer could vary depending on the disease model.
|
study
| 99.94 |
Yang et al. demonstrated that a brief treatment with α-GalCer in 7-week-old BWF1 mice suppressed lupus nephritis20. On the other hand, Zeng et al. reported contradictory results, namely that multiple injections of α-GalCer in 20-week-old BWF1 mice exacerbated lupus nephritis19. Regarding this point, a complex role for NKT cells in SLE, that is, a potential protective role before the onset of the disease and a potential pathogenic role after the establishment of the disease has been proposed16. Moreover, the age of the mice and the α-GalCer dose or administration interval may have influenced these conflicting results. However, we considered that the administration of α-GalCer to young mice was clinically unsuitable, because current international guidelines do not recommend the treatment of patients with mild lupus nephritis using immunosuppressive therapies38. We, therefore, began the α-GalCer treatment at 24 weeks of age, when mice may already develop mild renal disease.
|
study
| 99.94 |
Although some experimental agents have been used in SLE models with favorable outcomes22,39, in the present study we did not use such agents. Instead, we administered α-GalCer, a synthetic but specific ligand of NKT cells, to investigate the role of these cells in lupus nephritis. Definitive natural ligands of NKT cells have not been so far identified. However, considering that α-GalCer is a glycolipid antigen and that some microbes may be antigenic candidates for NKT cells40,41, we think that NKT cells are involved in lupus nephritis via the glycolipid metabolism or in response to microbes.
|
study
| 100.0 |
We have recently demonstrated that the function of NKT cells was enhanced in the absence of NK cells (Uchida et al. in submission). In the present study, we have demonstrated that IFN-γ levels produced by CpG-ODN-stimulated liver MNCs were significantly higher in the α-GalCer group. Moreover, because we have previously reported that NK cells produced IFN-γ after CpG-ODN stimulation24, we proposed a compensatory augmentation in the function of NK cells. Although the precise mechanisms remain to be elucidated, complementary roles may be shared between NKT and NK cells to avoid immunosuppressive states.
|
study
| 100.0 |
Our study has several limitations. It has been reported that BWF1 mice are superior to other SLE models in that they resemble the human disease, and therefore, are most commonly used42. Indeed, these mice spontaneously develop lupus nephritis-like renal lesions. However, the renal disease observed in these mice does not fully recapitulate that in humans; for example, hematuria was not observed in BWF1 mice despite proliferative glomerulonephritis. The role of NKT cells in the progression of human lupus nephritis also remains to be resolved using α-GalCer. This is partly because human NKT cells, which express T cell receptors encoded by the Vα24Jα18 and Vβ11 genes and respond to α-GalCer, are rare in the liver as well as other organs. In addition, decreased levels of human CD56+ T cells, which are not activated by α-GalCer but have been proposed as a functional counterpart for mouse NKT cells8,43,44, might be associated with high levels of serum IgG and anti-dsDNA Abs in patients with SLE14, suggesting that CD56+ T cells may ameliorate SLE. Therefore, it should be carefully evaluated in the future whether human NKT cells or CD56+ T cells are really involved in lupus nephritis.
|
study
| 99.94 |
In conclusion, the repeated administration of α-GalCer decreased the number of NKT cells and suppressed Th2 immune responses in these cells. This led to the suppression of B cell function and the autoimmune response, and ameliorated proteinuria and kidney pathology in experimental lupus nephritis. Therefore, we propose that NKT cells collaborate with B cells and play significant roles in the induction of lupus nephritis through their Th2 immune responses. Thus, it is important to focus on NKT cells to further investigate the precise pathogenesis of lupus nephritis.
|
study
| 99.94 |
The baseline urinary protein excretion of BWF1 mice was measured at 20 weeks of age, and the mice were assigned to either the α-GalCer group (n = 8), which was intraperitoneally injected with 2 μg/body of α-GalCer (Funakoshi, Tokyo, Japan) once a week for 4 weeks from 24 weeks of age, or the vehicle group (n = 10). The baseline urinary protein excretion was balanced between the two groups and amounted to less than 1 mg/day. The urinary protein excretion was thereafter measured periodically, and was considered to be positive when greater than 1 mg/day.
|
study
| 100.0 |
Mice were sacrificed under deep anesthesia at the end of the experiment (8 weeks after the last α-GalCer injection). All animal experiments were conducted in accordance with the National Defense Medical College guidelines for the care and use of laboratory animals in research. The study protocol (no. 15087) was approved by the Animal Ethics Committee of the National Defense Medical College.
|
other
| 99.9 |
Sections of formalin-fixed paraffin-embedded renal tissues were subjected to PAS staining, and 50 glomeruli were evaluated to assess glomeruli with immune complex deposits consisting of wire loop lesions and hyaline thrombi, sclerotic glomeruli including both globally and segmentally sclerotic glomeruli, and glomeruli with crescents.
|
study
| 99.94 |
Indirect immunoperoxidase staining for F4/80, CD3, or WT-1 was performed using rat anti-mouse F4/80 (Serotec, Oxford, UK), rabbit anti-human CD3 (Agilent, Santa Clara, CA), or rabbit anti-mouse WT-1 Abs (Santa Cruz Biotechnology Inc., Santa Cruz, CA), respectively.
|
other
| 99.9 |
Images of five non-overlapping areas from each section stained for F4/80 or CD3 were obtained with a digital camera at a magnification of 200×, and the percentages of the areas positive for each staining were measured using image analysis software (LuminaVision ver. 2.04, Mitani Corporation, Tokyo, Japan) and averaged. For WT-1 staining, 20 glomeruli were assessed, and the numbers of WT-1-positive cells per glomerulus were counted and averaged.
|
study
| 99.94 |
Extraction of total RNA, reverse transcription into complementary DNA, and the subsequent real-time PCR were all performed as previously described45. We used primer/probe sets of the TaqMan Gene Expression Assays for mouse CD11c, CD138, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), all obtained from Thermo Fisher Scientific (Waltham, MA). The relative amount of mRNA was calculated using the comparative Ct (∆∆Ct) method. All amplification products were normalized against GAPDH mRNA, which was amplified in the same reaction as an internal control.
|
study
| 99.94 |
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