In order to identify pure genomic transcriptional effects of 1,25(OH)2D3, we used VDR cistrome, transcriptome and open chromatin data, obtained from the human monocytic cell line THP-1, for a novel hierarchical analysis applying three bioinformatics approaches. We predicted 75.6% of all early 1,25(OH)2D3-responding (2.5 or 4 h) and 57.4% of the late differentially expressed genes (24 h) to be primary VDR target genes. VDR knockout led to a complete loss of 1,25(OH)2D3–induced genome-wide gene regulation. Thus, there was no indication of any VDR-independent non-genomic actions of 1,25(OH)2D3 modulating its transcriptional response. Among the predicted primary VDR target genes, 47 were coding for transcription factors and thus may mediate secondary 1,25(OH)2D3 responses. CEBPA and ETS1 ChIP-seq data and RNA-seq following CEBPA knockdown were used to validate the predicted regulation of secondary vitamin D target genes by both transcription factors. In conclusion, a directional network containing 47 partly novel primary VDR target transcription factors describes secondary responses in a highly complex vitamin D signaling cascade. The central transcription factor VDR is indispensable for all transcriptome-wide effects of the nuclear hormone.The transcription factor vitamin D receptor (VDR) is the high affinity nuclear target of the biologically active form of vitamin D The endocrine hormone 1,25(OH)2D3 binds with high affinity to the transcription factor vitamin D receptor (VDR), a member of the nuclear receptor superfamily1. Although particularly highly expressed in the small intestine and the kidney, VDR is also found in immune cells and many other organs, albeit at lower level2. This explains why VDR not only plays a role in the regulation of calcium and phosphorus homeostasis3, but also modulates other physiological functions, including innate and adaptive immune responses4. According to the canonical nuclear receptor signaling model, VDR binds as a heterodimer with the nuclear receptor retinoid X receptor (RXR) to response elements formed by a direct repeat of two hexameric sequence motifs spaced by three nucleotides (DR3)6. Numerous transcriptomic and epigenome-wide data obtained in human monocytic THP-1 cells were used to refine this model to better describe genomic vitamin D signaling involving possible modulation of the 3-dimensional chromatin structure as well as the action of pioneer factors like purine-rich box 1 (PU.1 or SPI1) and CCAAT/enhancer binding protein α (CEBPA)17.The lipophilic molecule 1α,25-dihydroxyvitamin D2D3 which are independent of transcriptional regulation. These effects appear to be cell type-specific and are mediated by kinases such as phospholipase A2 (PLA2), phospholipase C (PLC), protein kinase A (PKA), protein kinase C (PKC), Ca2+/calmodulin-dependent protein kinase (PKCaMII), mitogen-activated protein kinases (MAPK), Src tyrosine phosphorylation and phosphatidylinositol 3-kinase (PI3K)25. Reported physiological effects of non-genomic responses to 1,25(OH)2D3 comprise modulations of calcium uptake and secretion, intracellular pH, insulin secretion and cell migration, but their overall significance in vivo is unclear30. There is some controversy in the identification of the receptor(s) which convey 1,25(OH)2D3-induced non-genomic cellular function. Some reports state a completely VDR-independent propagation via a novel membrane receptor34. VDR has been found localized in membrane caveolae and to interact or synergize with the membrane receptor, Protein disulphide isomers family A member 3 39. It has also been reported that 1,25(OH)2D3 can induce non-genomic effects by binding to an alternative ligand binding pocket in the classical nuclear VDR29. Thus, there may be different parallel mechanisms for non-genomic actions of 1,25(OH)2D3, for some of which both VDR and PDIA3 are required43. It is generally agreed that non-genomic 1,25(OH)2D3 signaling facilitates its genomic regulation, for example by activating RXR or other transcription factors by phosphorylation44. In this study, we knocked down the VDR to determine whether any transcriptome-wide effects of 1,25(OH)2D3 can be observed in the absence of its nuclear receptor.In addition to the genomic actions, rapid non-genomic effects have also been described in response to 1,25(OH)45 and the TEPIC 2 framework for transcription factor binding site prediction46 to study primary and secondary effects of vitamin D.Further, we used the EpiRegio database of regulatory elements (REMs)47 is a well responding and physiologically meaningful model system for the investigation of 1,25(OH)2D3-triggered physiological processes, such as innate immunity and cellular growth50. The cells were grown in RPMI 1640 medium, supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 0.1 mg/ml streptomycin and 100 U/ml penicillin, and were kept at 37 °C in a humidified 95% air/5% CO2 incubator. Prior to mRNA or chromatin extraction, cells were grown overnight in phenol red-free medium supplemented with charcoal-stripped FCS and then treated with vehicle (0.1% ethanol (EtOH)) or 100 nM 1,25(OH)2D3 (Sigma-Aldrich).The human acute monocytic leukemia cell line THP-12 incubator.Lenti-X 293 T cells (Takara Bio) were grown in DMEM medium supplemented with 10% FCS, 0.1 mg/ml streptomycin and 100 U/ml penicillin and were kept at 37 °C in a humidified 95% air/5% COTHP-1 cells stably expressing type II CRISPR RNA-guided endonuclease Cas9 (Cas9), generated by lentiviral transduction with the plasmid lentiCas9-Blast  into THP-1 cells from DSMZ (ACC16), and the sequences for the non-targeting control guide RNAs (gRNAs) were kindly provided by Dr. Frank Schnuetgen .VDR gene have been designed using the Benchling life sciences R&D cloud51 and CHOPCHOP52. Sense and antisense oligonucleotides containing the 19 bp gRNA sequence plus the required overhangs matching the BsmBI-cut vector were ordered (Sigma-Aldrich) and annealed (Supplementary Table BsmBI (Esp3I) (Thermo Fisher) and T4 DNA ligase (Thermo Fisher) were used to clone the annealed gRNAs into the plasmid LentiGuide-Puro  with the Golden Gate assembly protocol53. Correct insertion of the gRNAs into the plasmid was verified by Sanger sequencing.Specific gRNAs targeting the Cell-free, lentiviral supernatants were produced by polyethylenimine (PEI)-based transient co-transfection of Lenti-X 293 T cells. Briefly, the pLentiGuide-Puro-gRNA vector, the lentiviral gag/pol packaging plasmid psPAX  and the envelope plasmid encoding the glycoprotein of vesicular stomatitis virus (VSV-G)  were transfected at a molar ratio of 3:1:1 by standard PEI transfection. 24 h and 48 h post transfection, two consecutive viral supernatants were harvested, cleared through 0.45-µm pore-size PVDF membrane filter (Millipore), combined and stored at − 80 °C.6 cells were pelleted and resuspended in 1 ml of the lentiviral particles. Polybrene (8 µg/ml) was added and the cells were centrifuged for 90 min with 1000 × g at room temperature. The supernatant was discarded, the cells resuspended in 1 ml full growth medium and incubated for 48–72 h. Then, the second spinoculation was carried out with 800 µl of lentivirus. After another 48–72 h incubation, puromycin (2 µg/ml) was added to select the transduced cells for 10–14 days.Prior to lentiviral transduction, the THP-1/Lenti-Cas9-Blast cells were re-selected with Blasticidine S hydrochloride (50 µg/ml) for 3 days. The transduction with the gRNA-expressing plasmids was performed in two consecutive spinoculations. Briefly, 0.2 × 10VDR knockout cells were re-selected with puromycin (2 µg/ml) for three days.Prior to each experiment for RNA extraction, the Cells were washed twice with phosphate-buffered saline (PBS) and lysed with Triton X-100 lysis buffer , 40 μg/ml phenylmethylsulfonyl fluoride). Cells were centrifuged for 10 min at full speed at 4 °C. The supernatant was transferred and after determination of protein concentration by the Bradford assay, equal amounts of protein were boiled in sample buffer and separated by SDS-PAGE (8%). The gels were blotted onto a nitrocellulose membrane and blocked in Rotiblock (Carl Roth). The anti-VDR antibody  and the β-actin antibody  were used in a 1:1000 dilution. The blots were developed with infrared-fluorescent-dye-conjugated secondary antibodies (LI-COR Biosciences) and detected with an Odyssey Classic system (LI-COR Biosciences).Total RNA was extracted using the Quick-RNA Miniprep Kit (Zymo Research) according to the manufacturer’s instructions. RNA quality was assessed on a Fragment Analyzer (Agilent). Four independent replicate experiments  were performed for RNA sequencing (RNA-seq).54. Differential gene expression was computed using DESeq2, which implements a negative binomial test over the reads in each two conditions 2D3-treated/solvent-treated or NTC control/VDR knockout), with standard parameters and an adjusted p-value cutoff of 0.0555. Information regarding which batch of THP-1 cells each sample originated from was provided to DESeq2 to correct for batch effects.RNA-seq libraries were prepared from 1 µg total RNA using the VAHTS Stranded mRNA-seq Library Prep Kit for Illumina V2  including poly(A) enrichment. Single end sequencing was conducted to 75 bp read length on the NextSeq500 platform using standard Illumina protocols resulting in roughly 20 M reads per library. The resulting raw reads were assessed for quality, adapter content and duplication rates with FastQC. Then, the high-quality reads were aligned to the reference genome (hg19) using Spliced Transcripts Alignment to a Reference (STAR) with the parameter –quantMode GeneCounts for gene-level quantification and otherwise default parameters.56 with some modifications. After treatment of 20 × 106 THP-1 cells with 100 nM 1,25(OH)2D3 for 24 h, nuclear proteins were cross-linked to genomic DNA by adding formaldehyde directly to the medium to a final concentration of 1% and incubating at room temperature for 10 min on a rocking platform. Cross-linking was stopped by adding glycine to a final concentration of 0.125 M and incubating at room temperature for 10 min on a rocking platform. The cells were collected by centrifugation and washed twice with ice cold PBS. The cell pellets were subsequently resuspended twice in 10 ml cell lysis buffer  and once in 10 ml nuclear lysis buffer . After two washes with cell lysis buffer, the chromatin pellet was resuspended in 700 µl of SDS lysis buffer  and the lysates were sonicated in a Bioruptor Plus (Diagenode) to result in DNA fragments of 200 to 500 bp. Cellular debris was removed by centrifugation. 340 µl aliquots of the lysate were diluted 1:5 in IP dilution buffer . 4 µg anti-ETS1 antibody , previously used by the ENCODE project (data available at Gene Expression Omnibus (GEO)57 at GSE32465) were coated to 60 µl Dynabeads Protein G (Invitrogen) in an overnight incubation at 4 °C. The pre-formed bead-antibody complexes were then washed twice with beads wash buffer  and added to the chromatin aliquots. The samples were incubated for overnight at 4 °C on a rotating wheel to form and collect the immuno-complexes. The beads were washed sequentially for 5 min on a rotating wheel with 1 ml of the following buffers, each: twice cell lysis buffer, once high salt buffer , once ChIP wash buffer  and twice 1 ml TE buffer . Then, the immune complexes were eluted using 250 µl ChIP elution buffer  at 37 °C for 30 min with rotation. The elution was repeated with a 10 min rotation and the supernatants were combined. The immune complexes were reverse cross-linked at 50 °C for 2 h in the presence of proteinase K (Fermentas) in a final concentration of 40 µg/ml. The genomic DNA was isolated with the ChIP DNA Clean&Concentrator Kit (Zymo Research). Two biological replicates have been prepared for the ETS1 sequencing (ChIP-seq).ChIP assays were performed as described by Zhang et al58 with the following essential command line arguments: macs2 callpeak –bw 150 –keep-dup 1 –g hs –qvalue = 0.01 –m 5 50 –bdg. Peaks are declared when enriched regions, compared with the corresponding loci in the input, are found. Otherwise, default parameters were used. ETS1 ChIP-seq raw data are available at GEO at GSE157209.2–10 ng of each ChIP DNA template was used for library preparation with the NEBNext Ultra II kit (New England Biolabs) and libraries were sequenced at 50 bp read length on a HiSeq2000 system using standard Illumina protocols at the Gene Core of the EMBL . ChIP-seq data was aligned with the human reference genome version hg19 using Bowtie software version 1.1.1 with the following essential command line arguments: bowtie -n 1 -m 1 -k 1 -e 70 –best. The aligned input and ETS1 reads were converted to sorted BAM format using samtools and, after merging the read sets per sample, converted to TDF format using igvtools, in order to allow efficient visualization in the Integrative Genomics Viewer (IGV) genome browser. Statistically significant ChIP-seq peaks were identified using Model-based Analysis of ChIP-Seq data (MACS) version 2.11. CEBPA ChIP-seq data can be accessed under GSE11955613 and H3K27ac ChIP-seq under GSE10785112. Formaldehyde-assisted isolation of regulatory elements sequencing (FAIRE-seq) data in THP-1 cells after 24 h of 100 nM 1,25(OH)2D3 treatment is available from the same source under the accessions GSE69297 and GSE69303, originally published by Seuter et al.15. RNA-seq time course data in THP-1 cells with 2.5 h, 4 h and 24 h of 100 nM 1,25(OH)2D3 treatment is accessible at the accession GSE6930315.VDR ChIP-seq data in THP-1 cells is publicly available at NCBI GEO under the accession GSE89431, originally published by Neme et al46. In order to make use of the VDR ChIP-seq data to identify direct VDR target genes, we used (i) TEPIC 2 to associate VDR ChIP-seq peaks to genes by considering a window of 50 kb centered at the transcription start site (TSS) of genes. Further, (ii) we overlapped VDR ChIP-seq peaks with annotated REMs of the EpiRegio database45. REMs in EpiRegio are linked to genes. Each gene that had at least one REM that was linked to it and overlapped a VDR ChIP-seq peak was considered a direct VDR target.TEPIC (version 2.2) is a universal software for the annotation of genes and integration of epigenomic, ChIP-seq and TF motif data2D3 treatment and looked for predicted VDR binding sites using position weight matrices of VDR or RXRA::VDR obtained from merged JASPAR 2020 59, HOCOMOCO 60 and Kellis-ENCODE61 motif sets included in TEPIC 2.2. Transcription factor binding affinities to regions of open chromatin are calculated using TRAP. Predicted VDR sites were assigned to genes if they were present in a window of 50 kb around the TSS of a gene. Only affinities meeting a threshold determined by user-supplied random sequences were used to assign transcription factors to genes. Calculated affinities are subject to exponential decay depending on the distance of the predicted transcription factor binding site from the TSS of the respective gene.Finally, (iii) we made use of FAIRE-seq data in THP-1 cells after 1,25(OH)2D3 treatment measured with RNA-seq.The direct target genes obtained by the three approaches above were intersected with sets of DEGs after 1,25(OH)2D3 treatment. Transcription factor binding sites were predicted using position specific energy matrices derived from JASPAR 202059, HOmo sapiens COmprehensive MOdel COllection (HOCOMOCO)60 and Kellis ENCODE61 motif databases, which are part of the TEPIC 2 repository.We used TEPIC 2.2 to annotate open-chromatin regions detected with FAIRE-seq in THP-1 cells after 24 h of 1,25(OH)2D3 treatment, which were not identified as direct VDR target genes. DYNAMITE used transcription factor-gene scores calculated by TEPIC 2 from the FAIRE-seq data before and after treatment for a set of 720 transcription factors. These scores were used as features for a sparse logistic regression classifier that predicts up- and down-regulated DEGs. The classifier learns a coefficient for each transcription factor included in the analysis, which describes the importance of the respective transcription factor to the modulation of gene expression in the dataset. We normalized the coefficients obtained from the classifier to the range .The DYNAMITE workflow of TEPIC 2, was used to learn the importance of individual transcription factors to gene expression changes of the subset of DEGs in THP-1 cells after 24 h of 100 nM 1,25(OH)igraph package in R (version 4.0.0). Edges were added between VDR and transcription factors predicted to be primary 1,25(OH)2D3 targets. Further edges were added between early-induced transcription factors and the rest of the induced transcription factors, if the early-induced transcription factor was predicted to bind at the gene locus (TSS + /- 25 kb) of the second transcription factor at an affinity greater than a given threshold by TEPIC 2. The network was plotted using the ggraph package in R.The transcription factor network was constructed using the 62. Significantly overrepresented pathways were those with a Benjamini–Hochberg adjusted P < 0.05.Pathway enrichment analysis was performed using the R (4.0.2) package ReactomePA (1.32.0)2D3 for 2.5, 4 or 24 h, the total number of DEGs had been reported by Neme et al.11 as 3650. Among these were 19 non-annotated transcripts, which were excluded for this study. Of the remaining 3631 annotated genes, 513 genes were differentially expressed after 2.5 or 4 h of stimulation and as such were classified as early 1,25(OH)2D3-responsive genes. No genes were transiently differentially expressed. Commonly, early responding genes are considered directly regulated by a transcription factor. Thus, in our previous studies in THP-1 cells, we have classified genes as primary vitamin D targets if they were differentially expressed at early time points of 1,25(OH)2D3 treatment, whereas those differentially expressed only at a late time point (24 h) were classified as secondary target genes63. According to this classification, the vast majority of all differentially expressed genes (DEGs) were secondary vitamin D target genes , while only 517 genes were early responding primary targets. However, since there are many VDR-bound regions (ChIP-seq) close to many of these so-called secondary vitamin D target genes, it seems likely that these genes can be regulated by VDR-bound enhancer elements. Hence, a better method to identify direct transcription factor targets was needed.In THP-1 cells treated with 1,25(OH)65. Thus, in the present study, we combined three different approaches to predict primary vitamin D target genes from 1,25(OH)2D3-responding genes  used REMs linked to genes from the EpiRegio database45. EpiRegio holds elements which have been identified by correlations between chromatin accessibility and gene expression at loci in a window of gene body ± 100 kb using data from the BLUEPRINT66 and the Roadmap consortia67. VDR ChIP-seq peaks overlapping with an REM from EpiRegio were linked to DEGs45. In the third approach (iii), the TEPIC 2 framework was used to search for VDR binding motifs obtained from diverse motif databases (see Methods) overlapping FAIRE-seq peaks that were located in a window of ± 25 kb around the TSS of 1,25(OH)2D3 DEGs. TEPIC 2 is a method that computes transcription factor gene scores based on position specific energy matrices of transcription factors and the signal of an open-chromatin assay46. By limiting the search space around genes to nearby regions of open chromatin, the prediction of contextually important transcription factor binding is more likely than using a continuous search space of the gene region without epigenetic information.However, linking regulatory regions to target genes is not easy and each method has its limitations2D3-responsive genes (75.6%) were predicted to be primary VDR targets by any of the three methods. The remaining 130 genes (24.4%) were classified as non-primary , heparin binding EGF like growth factor (HBEGF) and ninjurin 1 (NINJ1)69.According to the primary VDR target gene criteria set out above, 383 early-induced 1,25(OH)2D3 treatment, which were not differentially expressed at either 2.5 h or 4 h, and as such were classified as late 1,25(OH)2D3-responsive genes. Of these late-responding genes, 1,788 (57.4%) were classified as primary VDR targets by the aforementioned criteria, leaving 1,329 (42.6%) of the late-responding 1,25(OH)2D3-regulated genes as secondary vitamin D target genes Of the early induced vitamin D target genes, 61.1% (234 of 383) of the primary VDR target gene predictions have been confirmed by at least two of the three approaches, 79 genes (20.6%) by all three. 100 genes (26.1%) were assigned primary early VDR targets only based on window-based VDR binding  were predicted to be primary VDR target genes by all three approaches, and 42.4%  by at least two of the three methods. There were 368 genes (20.6%) assigned as primary VDR targets by the combination of transcription factor binding site prediction using TEPIC 2 in conjunction with 1,25(OH)2D3-stimulated FAIRE-seq data and a window based VDR peak to gene assignment and another 636 (35.6%) by the VDR association alone 2D3-responding and 57.4% of the late DEGs were predicted to be primary VDR target genes.Taken together, using three bioinformatics approaches including VDR ChIP-seq data, VDR motif search in open chromatin regions and REMs identified from multiple published genome-wide datasets, 75.6% of all early 1,25(OH)2D3-regulated gene expression. The knockout was achieved by lentiviral clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated gene editing. Sufficient knockout on protein level was confirmed by Western blot for knockout lines generated with four different gRNAs 2D3 were 109 and 1,341, respectively 2D3 exposure 2D3. Three observations from the differential expression analysis between VDR knockout and control cells confirmed the complete dependence of 1,25(OH)2D3 regulation on the presence of VDR: (i) in ligand-treated cells similar numbers of genes were differentially expressed as after stimulation with 1,25(OH)2D3 in control cells 2D3-stimulated cells for both time points  demonstrates that for most genes the effect of 1,25(OH)2D3 on mRNA expression is not only reduced but completely lost upon VDR depletion  and potassium voltage-gated channel modifier subfamily F member 1 (KCNF1), the late primary VDR targets peroxisome proliferator activated receptor gamma (PPARG) and C-X-C motif chemokine ligand 8 ) and the late secondary target leukocyte receptor tyrosine kinase (LTK) 2D3–induced gene regulation in monocytic THP-1 cells, i.e., transcriptome-wide effects of vitamin D were entirely mediated by VDR.In summary, VDR knockout led to a complete loss of 1,25(OH)70. To globally identify the indirect mechanisms by which the 1,517 non-primary vitamin D target genes (41.8%) were regulated, we screened the primary VDR target genes for transcription factor genes and identified 47 among them. A subset of these genes were early vitamin D target genes, including the pioneer factors CEBPA and ETS proto-oncogene 1, transcription factor (ETS1) as well as BCL6 and NFE2. The remaining 34 transcription factor genes only showed upregulation after 24 h of 1,25(OH)2D3 treatment. Among these were genes such as nuclear factor I A (NFIA), the nuclear receptor PPARG and cut like homeobox 1 (CUX1) ) in regions of open chromatin as determined by VDR ChIP-seq and FAIRE-seq, respectively. This supported the prediction that these genes are direct VDR targets  were predicted to be primary VDR targets according to the criteria and methods used in this study. For the physiological impact of vitamin D signaling, secondary responses are as important as primary effects. Previously, the four transcription factors B-cell CLL/lymphoma 6 (BCL6), nuclear factor erythroid 2 (NFE2), E74-like ETS transcription factor 4 (ELF4) and POU class 4 homeobox 2 (POU4F2) have been shown to mediate secondary effects of vitamin D2D3 and being predicted to be primary VDR targets 47 were coding for transcription factors.Taken together, among the genes differentially expressed by 1,25(OH)2D3 treatment was characterized. Factors were prioritized that could explain secondary 1,25(OH)2D3-responsive genes, for which no direct VDR regulation could be established. For this purpose the DYNAMITE approach (see Methods) was used, in which motif based transcription factor binding prediction identifies meaningful transcription factors for a set of up- and down-regulated genes46. There were 22 transcription factors predicted to be of importance for the regulation of the secondary vitamin D target genes, amongst them CUX1, interferon regulatory factor 5 (IRF5), PPARG, NFE2 and ETS1 TS1 Fig. .Figure 42D3, the TEPIC 2 framework was used to predict which of the late (24 h only) 1,25(OH)2D3-responsive genes are bound by the 47 primary vitamin D target transcription factors  and bound more often to primary than to secondary vitamin D target genes , membrane spanning 4-domains A14 (MS4A14), membrane spanning 4-domains A4A (MS4A4) and LTK. The ChIP-seq data confirmed strong ETS1 binding to a region close to the TSS of COX7A2L located in open chromatin and marked with the enhancer specific H3K27ac histone modification. CEBPA co-located to the same region and bound more strongly to another region in the locus  was also confirmed by the CEBPA ChIP-seq data he late 2 h only 1he late 2 h only 1he late 2 h only 1he late 2 h only 1he late 2 h only 1In order to determine whether the subsets of CEBPA or ETS1 bound vitamin D target genes represent different physiological functions, a pathway enrichment analysis was performed. No pathways were significantly connected to the vitamin D targets associated by CEBPA 2D3 treatment, at the locus of the late 1,25(OH)2D3-induced IRF5 gene. VDR associated strongly close to the TSS of the gene, to an intergenic enhancer and to an enhancer approximately 25 kb upstream of the TSS. CEBPA and ETS1 bound to at least two enhancers each  and ETS1 and the predicted binding of VDR and ETS1 to the gene coding for the general transcription factor TATA-box binding protein (TBP) were confirmed by the ChIP-seq data CEBPA verified that the predicted binding of CEBPA correlated with functional effects13. Of the vitamin D target genes identified from the wildtype THP-1 RNA-seq, 1,256 have been predicted to bind CEBPA. More than half of these (734) were also differentially expressed by 1,25(OH)2D3 in control or CEBPA knockdown cells, or both 2D3 only in the absence of CEBPA. A rather high number of 448 vitamin D target genes were conserved vitamin D targets irrespective of the CEBPA silencing. However, as can be seen from the heatmap displaying the induction 2D3) of the top 50 of these genes, for most of them the CEBPA knockdown reduced the response to the VDR ligand  and GLIS family zinc finger 3 (GLIS3) 2D3 following CEBPA depletion. A directional network containing 47 primary VDR target transcription factors indicated a high complexity of vitamin D signaling and suggests novel factors.In summary, CEBPA and ETS1 ChIP-seq data validated the predicted association of these transcription factors to secondary vitamin D target genes. Approximately half of the vitamin D target genes predicted to bind CEBPA showed lost, gained or modulated response to 1,25(OH)2D3-regulated transcriptome in monocytic THP-1 cells to predict primary VDR target genes on the basis of RNA-seq, VDR ChIP-seq, FAIRE-seq, VDR motif analysis and the EpiRegio database of REMs. Our analysis indicated that the classification of 1,25(OH)2D3 DEGs into primary and secondary vitamin D target genes just by time points is an oversimplification. A high proportion (72.1%) of all early (2.5 or 4 h) 1,25(OH)2D3-regulated genes were predicted to be direct VDR targets, which may not seem surprising as it fits well with the time point-based classification. However, from the late-responding genes (24 h), 56.3% were assigned primary targets by our approach, which is a high ratio considering that these genes are usually considered secondary target genes due to their slow response. From the three approaches we applied, VDR ChIP-seq peaks were assigned to the highest numbers of vitamin D-responsive genes. Among them were many genes for which a VDR binding site was not predicted by TEPIC 2. This is likely due to imperfect motif models of the complex VDR binding profile, or the binding of VDR with co-factors resulting in the presence of VDR in regions where no canonical VDR binding motif is present. It is also in line with the observation that only 47.6% of the VDR cistrome carried a DR3-type VDR motif11.In this study, we used a hierarchical analysis of the 1,25(OH)2D3-responding genes can be a secondary target gene and thus our predictions have been too stringent. Inducible transcription factors lead to bursts of transcription and the bursting period commonly lasts several hours during which several transcriptional events take place75. For a secondary response, after the several hours of primary transcriptional activation the transcription factor has to be translated and then may induce a second transcriptional activation on its own. Thus, it is very unlikely that secondary responses can be detected in the time frame of 4 h. However, no matter which stringency we apply, a major outcome of this study is that a significant number of all DEGs that are unique to the 24 h time point of 1,25(OH)2D3 stimulation are primary VDR target genes. There is some inaccuracy in all RNA-seq data, which we have observed for example for the CXCL8 (IL8) gene. According to both RNA-seq datasets used in this study, the gene is not significantly regulated after 2.5 or 4 h stimulation with 1,25(OH)2D3 and was therefore classified as late response gene. However, in a previous report the gene has been shown to be significantly upregulated by 1,25(OH)2D3 in THP-1 cells already after 1 h by RT-qPCR76. If the same were true for many of the late responding genes, the discordance between primary target gene predictions and time point-based classification would be reduced. A gene may be directly regulated by VDR, but mRNA accumulation may be low if either the transcriptional burst is small or if mRNA stability is low. In such cases, the detection by RNA-seq may only be possible with very high sequencing depth. Assuming that not only technical reasons prevented the detection of weak mRNA levels accurately, our data indicate that for some primary vitamin D target genes the transcriptional response is delayed. This could be due to differences in co-activator recruitment or co-repressor disposal or to lower local chromatin accessibility. Also, the number and position of VDR sites in the gene locus may play a role. It has to be noted, though, that there is a rather big gap between the 4 h and the 24 h time-point and some genes may already be significantly regulated after 6 h or 8 h and thus may not be that late-responding after all. In contrast to the TEPIC 2-based approaches, the REMs in the EpiRegio database have been determined in a window of + /100 kb around and including the gene body. In spite of that less stringent window size, there are rather few primary target gene predictions made by this approach that have not been confirmed by at least one of the other two approaches. The REMs have been determined from large numbers of genome-wide datasets, which highly increased the statistical power of this method and rendered it more quantitative.Increasing the stringency by classifying only those DEGs as primary, which have been predicted by at least two of the three prediction methods reduced the ratio of primary target genes to 45.6% and 24.4% for early and late responding genes, respectively. Thus, this would indicate that 54.4% of the early regulated genes might in fact be secondary vitamin D target genes. The stringency of the prediction methods greatly depends on the window sizes that have been applied. For the FAIRE-seq motif and ChIP-seq approaches performed in TEPIC 2, we used a window of ± 25 kb around TSSs of the genes. Whilst we were aware that enhancers could be more distal from target genes than the threshold suggested by that window size, an increased window size would have led to many more false positive primary target gene predictions. On the other hand, it could be argued that none of the early 1,25(OH)VDR gene completely abolished 1,25(OH)2D3-induced transcriptomic changes as measured by RNA-seq. This on the one hand validated the identified vitamin D target genes, which could no longer be regulated by 1,25(OH)2D3 if its nuclear receptor VDR was not present to act as a ligand-activated transcription factor. It also indicated that even weakly up- or downregulated genes are indeed true vitamin D target genes and presumably their regulation plays a biological role. On the other hand, in light of reports about non-genomic effects of 1,25(OH)2D3 which have been described to also alter the activity of transcription factors like RXR and thus crosstalk with transcriptional regulation by its heterodimerization partner VDR44, it is surprising that there are no genes differentially expressed by 1,25(OH)2D3 in the VDR knockout cells. This contradicts reports about such rapid 1,25(OH)2D3 actions being independently of VDR mediated by an alternative receptor as PDIA339 and indicates that in the monocytic THP-1 cells such rapid effects of 1,25(OH)2D3 either involve the classical VDR by binding to its alternative binding pocket29, or are not present at all.Knockout of the 2D3 and which were predicted to be responsible for secondary effects of the active form of vitamin D. Each of these transcription factors was predicted to bind at many vitamin D-responsive genes, with some linked to thousands of DEGs. Among these were primary and secondary target genes, some of these being transcription factors as well. Using ChIP-seq data for the pioneer factors ETS1 and CEBPA, we could validate some of the predicted associations to vitamin D target gene loci. The myeloid lineage-determining transcription factor CEBPA is known to co-locate with VDR at more than 5,000 regions and, as an additional layer of complexity, at ≈1,500 sites CEBPA binding was modulated by the VDR ligand13. CEBPA silencing affected both the basal expression levels and the induction by 1,25(OH)2D3 of more than half of all strongly regulated vitamin D target genes. The latter represents both the pioneering function of the transcription factor, where CEBPA co-locates with VDR at primary VDR target genes, and the regulation of genes by CEBPA that are no direct VDR targets. This regulation was increased by the 1,25(OH)2D3-induced upregulation of CEBPA expression and by increased binding of CEBPA to some of its binding sites. We could correlate the predicted binding of CEBPA to a few hundred vitamin D target genes to functional changes by the CEBPA knockdown. This confirmed about a quarter (based on significant changes of the vitamin D response) to half (including non-significant changes) of our predictions for this transcription factor. There are additionally more than 1,000 genes that either lost or gained response to 1,25(OH)2D3 when depleting CEBPA, but were not predicted to bind CEBPA. These genes may represent secondary CEBPA target genes, or they were not predicted as CEBPA-bound due to imperfect CEBPA motif models or indirect DNA-association of CEBPA in a complex with other transcription factors. Previously, VDR and CEBPA have been shown to be tightly co-expressed in a module that is up-regulated in monocytes compared to cells from other hematopoietic lineages77. According to that study many transcription factors can be assembled into densely interconnected transcriptional circuits, which can provide a mechanism for robust gene regulation. Our results are inline with these findings and may be an explanation why monocytic cells are much more responsive to 1,25(OH)2D3 than some epithelial cell-types even though VDR expression is similarly high in both. Most probably other modules are highly expressed in these epithelial cells and if certain co-activators, chromatin-modifiers or vitamin D-regulated transcription factors are not in the expressed modules together with VDR the responsiveness to the VDR ligand will be lower. We presented a VDR-centered directional network of 47 VDR targeted transcription factors to describe the complexity of global vitamin D signaling in THP-1 cells. Most probably this network is cell-type specific and will contain partly other transcription factors in other cells. The often very strong increase in mRNA expression levels of primary vitamin D target genes from short to long time-points of ligand stimulation can partly be explained by such additional secondary 1,25(OH)2D3 responses.Another major result of this study is that there are 47 transcription factors whose expression was regulated by 1,25(OH)2D3 actions and to mechanistically characterize secondary effects of the VDR ligand in THP-1 cells. We predicted a high proportion of late-responding DEGs, that commonly are considered secondary target genes, to be in fact primary VDR targets. The remaining secondary VDR target genes appear to be regulated by a network of 47 transcription factors whose expression is directly regulated by 1,25(OH)2D3. This network is VDR-centered as we could show that all transcriptome-wide effects of the VDR ligand depended strictly on the presence of VDR. Thus, any non-genomic effects of 1,25(OH)2D3 that may modulate its transcriptional function, have to be mediated by VDR as well. The main findings from this study refined our previously published model of vitamin D signaling78 of which an adapted version is depicted in Supplementary Figure In summary, we applied a novel hierarchical approach to distinguish primary from secondary 1,25(OH)Supplementary files.Supplementary Table 2Supplementary Table 3Supplementary Table 4


CYP27B1 gene mutations in two large pedigrees.Vitamin D-dependent rickets type IA (VDDR-IA) is a rare autosomal recessive disorder characterized by the early onset of severe rickets. The objectives of this study were twofold: (1) to analyze the clinical characteristics and therapy of two patients with VDDR-IA from two separate Chinese families, and (2) investigate the 2D3] of the two patients and their respective families were measured by ELISA and blood samples from both families was obtained for CYP27B1 gene sequence.Medical history, clinical manifestations, physical examination, radiological findings and laboratory data were analyzed from two patients with VDDR-IA. Serum 1, 25-dihydroxyvitamin D [1, 25-(OH)3. However, serum 1,25-(OH)2D3 level were low in the patients but normal in their family members. Genetic sequence identified two patients were homozygous for a duplication mutation in exon 8 of CYP27B1 gene . After treating with calcitriol and calcium, there was biochemical improvement with normalization of serum calcium and phosphorus, and radiographic evidence of compensatory skeletal mineralization. One patient developed nephrocalcinosis during follow-up.Two patients had typical manifestations and radiological evidence of rickets. Laboratory data showed hypocalcaemia and hypophosphataemia, along with high levels of serum alkaline phosphatase, parathyroid hormone and 25-hydroxyvitamin DCYP27B1 in two large pedigrees, and compared the clinical characteristics and individual therapy of two affected patients. Additionally, our experience further supports the notion that nephrocalcinosis can occur even on standard doses of calcitriol and oral calcium, and normal level of serum calcium, phosphorus, PTH and 25-(OH)D3.This study identified a recurrent seven-nucleotide insertion of  The second rate-limiting step , acting through a special vitamin D receptor, the biological actions of 1, 25-(OH)2D3 encompass the regulation of calcium homeostasis, cellular differentiation, and immune function [Vitamin D consists of a group of biologically inactive, fat-soluble prohormones which exist in two major forms: ergocalciferol (vitamin Dsunlight . Both fovitamin D is derivCYP27B1), located on 12q13.3 [3 and decreased serum 1, 25-(OH)2D3 [CYP27B1 gene have been identified in patients from different ethnic groups [CYP27B1 mutation is a missense mutation, representing more than half of those reports [Vitamin D-dependent rickets type IA (VDDR-IA), also referred to as pseudo-vitamin D deficiency rickets, was first identified by Prader et al.  in 1961.-(OH)2D3 . To datec groups –12. ThesCYP27B1 gene in two patients and their family members.In the present study, we investigated two patients with VDDR-IA from two separate Chinese families and identified a previously reported mutation of This study was approved by the Ethics Committee of the Fuzhou Children's Hospital of Fujian, and informed consent was obtained from the participants’ legal guardians before participating in the study. The two patients are from two Chinese families of Han ethnicity, both of whom lived in Putian City, Fujian Province.2D3 level, normal serum phosphorus, elevated ALP, 25-(OH)D3 and PTH levels (Table Patient 1 was a 33-month old girl and was the first child of consanguineous parents (first-degree cousins). Her mother’s pregnancy was uneventful. She was born at a full term with a normal birth weight and height. She grew poorly with delayed motor skills, sitting unsupported at the age of 10 months. By 14 months, she could stand with support and had developed a lower limb “O” shaped deformity. She presented with hypocalcaemic seizures at 18 months. Calcitriol 0.25 μg/day and calcium 500 mg/day divided three times a day orally were therapeutic for 15 months. Physical examination showed her height was 76 cm (− 4.84 SD), and weight was 9 kg. Her hair was normal. She had pigeon breast, tibial bowing and widened metaphyses of the wrists and ankles. Laboratory data showed hypocalcemia, hypophosphataemia, low serum 1,25-(OH)2D3 level, normal serum phosphorus, elevated ALP, 25-(OH)D3 and PTH levels  and recurrent respiratory infections. She could stand with support, but with evident muscle weakness at 20 months and reportedly irritable. Her height was 68.3 cm (− 5.39 SD), and weight was 7.5 kg. She had late fontanel closure and anterior fontanelle was large (1 cm × 1 cm). She did not have alopecia. There were 18 teeth present and no odontodysplasia. Her maxillary second primary molars were unerupted. Rachitic rosary, eversion of the costal margin, scoliosis and widened metaphyses of the wrists and knees were evident. Laboratory data showed hypocalcemia, hypophosphataemia, low serum 1,25-(OH)3 were detected by Electrochemical luminescence method , and 1,25-(OH)2D3 were detected by Quantikine Elisa kit .Serum calcium was detected by CPC methods , phosphorus was detected by phosphomolybdate methods , ALP was detected by colorimetric method , PTH was detected by Chemiluminescence method , 25-(OH)DRadiographic studies were performed in the department of radiology at the Fuzhou Children’s Hospital of Fujian Medical University. Plain X-ray of the lower extremities was performed to detect bone deformities.After informed consent, peripheral venous blood from both two patients and their family members was taken. In addition, we obtained blood samples from 101 healthy persons of Han ethnicity  as control, all of whom were long-term residents living in Putian City, Fujian Province.CYP27B1 were amplified using 2 X PCR MasterMix Taq polymerase  by PCR from 100 ng of genomic DNA, and primers were designed as published previously [CYP27B1 gene reference sequence (NC_000012.11 and NM_000785.3) to identify the underlying molecular defect VDDR-IA.Genomic DNA from peripheral blood leucocytes was isolated using the QIAamp Blood DNA Mini Kit  following the manufacturer’s instructions. All nine exons and intron–exon boundaries of 3 and 1, 25-(OH)2 D3 were measured at each clinic visit.In patients 1 and 2, clinical data was collected before and after treatment with calcitriol and calcium. After starting calcitriol treatment, serum levels of calcium, phosphate, ALP and PTH as well as 25-(OH) D3, and low serum 1, 25-(OH)2D3 level. Both patients had normal renal function, which excludes congenital hypophosphatemic rickets characterized by bone mineralization disturbances related to hypophosphatemia secondary to proximal renal loss of phosphate. The diagnosis of 25-hydroxyvitamin D-1α-hydroxylase deficiency was established in each patient based on their clinical and biochemical features.Both patients presented with failure to thrive, muscle weakness and typical phenotypic features of rickets in early childhood. Laboratory tests showed hypocalcaemia, hypophosphataemia, high levels of serum ALP, PTH and 25-(OH) DCYP27B1 gene (c.1319_1325dupCCCACCC/p.Phe443Profs * 24). No identical mutations were detected in the 101 unrelated control samples (data not shown).Genetic sequence identified two patients were homozygous for a duplication mutation in exon 8 of Family 1 Fig. : The proFamily 2 Fig. : Identic3 was achieved  and calcium 600 mg/day (68 mg/kg/day) orally, divided three times per day. After 1 month of treatment, serum phosphorus normalized with a marked reduction of serum PTH, however, the hypocalcaemia persisted  and calcium 300 mg/day (40 mg/kg/day) orally, divided three times a day. After 2 months of treatment, she was 2.7 cm taller and 1 kg heavier. Despite this therapy, hypocalcaemia and hypophosphataemia persisted D rickets . Hypophoets [2D3 , 16. Our3 to 1,25(OH)2 D3 [3 and concomitant low levels of 1, 25-(OH)2D3 (4). Vitamin D-dependent rickets type II is caused by mutations in the vitamin D receptor gene. Alopecia occurs in approximately two-thirds of cases, consequent to a loss of vitamin D receptor activity within keratinocytes [2D3.Vitamin D dependent rickets can have two different etiologies. VDDR-IA is caused by 1α-hydroxylase deficiency which impairs the conversion of 25-(OH)D(OH)2 D3 . It is c(OH)2 D3 . The chainocytes . In starCYP27B1 gene.In our two patients, the aforementioned clinical and biochemical findings were consistent with the VDDR-IA. However, diagnosis should be confirmed by mutational analysis of the CYP27B1 gene found the same mutation in both patients: homozygous for the seven-nucleotide duplication in exon 8 of CYP27B1 , which is the first report in Chinese patients. Their parents were heterozygous carriers. This mutation changes the reading frame downstream of codon 442 and creates a premature stop signal at codon 466 (Phe443Profs * 24), resulting in complete loss of enzymatic activity. To date, 11 Chinese patients with VDDR-IA have been reported [Sequence analysis of the reported , 20. AllCYP27B1 gene in this healthy population did not find c.1319_1325dupCCCACCC or other mutations. Hence, this mutation is not a local common variant, rather suggesting a remote consanguinity in these families.In 1998, Wang et al.  investig3.Due to 1α-hydroxylase deficiency, alphacalcidol/calcitriol and calcium supplementation is needed to correct the biochemical abnormalities and skeletal lesions . Both pa2D3 deficiency and concomitant low serum calcium concentrations, bone fractures are not generally a component of VDDR-1A [Both of our patients presented with similar features  and similar biochemical findings , 12, 20. VDDR-1A , 25.CYP27B1 mutation of c.1319_1325dupCCCACCC. Following calcitriol and treatment, she improved spontaneously at 11 years of age. Interestingly, she did not require treatment to maintain normal serum calcium and 1, 25-(OH)2D3. Another patient with mild features caused by the same mutation was reported in a series by Wang et al. [CYP27B1 knockout mice can convert 25-(OH)D3 to 1, 25-(OH)2D3 [Durmaz et al.  reportedg et al. . Remissi-(OH)2D3 .CYP27B1, c.1319_1325dupCCCACCC, p.Phe443Profs * 24. This is the first report of homozygous patients with this seven-nucleotide duplication in Chinese patients, and the first detailed study of the mutation in two large Chinese pedigrees in the same small low-populated city. Furthermore, the comparison and analysis of the clinical characteristics and individual therapy of two patients suggests that high-dose calcitriol is required for those with disturbed severely disturbed calcium homeostasis.In this study, we reported two patients with VDDR-IA with typical clinical manifestations, and genetic sequence analysis found both patients homozygous for the same gene mutation of


S100 calcium binding protein A9 (S100A9) is a pro-inflammatory alarmin associated with several inflammation-related diseases. However, the role of S100A9 in lung injury in sepsis has not been fully investigated. Therefore, the present study aimed to determine the role of S100A9 in a lipopolysaccharide (LPS)-induced lung injury murine model and its underlying molecular mechanisms.−/− mice. To investigate the effects of S100A9 blockade, mice were treated with a specific inhibitor of S100A9. Subsequently, lung injury and inflammation were evaluated by histology and enzyme‑linked immunosorbent assay (ELISA), respectively. Furthermore, western blot analysis and RT-qPCR were carried out to investigate the molecular mechanisms underlying the effects of S100A9.LPS was utilized to induce sepsis and lung injury in C57BL/6 or NOD-like receptor family pyrin domain containing 3 (NLRP3)−/− mice. In vitro, S100A9 downregulation mitigated LPS-induced inflammation. Interestingly, these effects were blunted by NLRP3 overexpression.S100A9 was upregulated in the lung tissues of LPS-treated mice. However, inhibition of S100A9 alleviated LPS-induced lung injury. Additionally, S100A9 blockade also attenuated the inflammatory responses and apoptosis in the lungs of LPS-challenged mice. Furthermore, the increased expression of NLRP3 was also suppressed by S100A9 blockade, while S100A9 blockade had no effect on NLRP3The results of the current study suggested that inhibition of S100A9 could protect against LPS-induced lung injury via inhibiting the NLRP3 pathway. Therefore, S100A9 blockade could be considered as a novel therapeutic strategy for lung injury in sepsis. Sepsis is a fatal disease, characterized by multiple organ failure. The lung is the most vulnerable organ during sepsis . SeriousTrauma and infection are the main causes of sepsis and related lung injury. Both conditions can trigger excessive inflammatory responses in the lung . A growiNOD-like receptor family pyrin domain containing 3 (NLRP3) is one of the most important mediators of inflammation . Upon in−/− mice with C57BL/6 background were obtained from the Key Laboratory of Child Development and Diseases of Children’s Hospital of the Chongqing Medical University. Mice were housed at a temperature of 21–22 °C and relative humidity of 50% with a 12-h light/dark cycle. Animals were treated with 5 mg/kg LPS in sterile saline to induce lung injury, as described previously [All animal experimental procedures were approved by the Animal Care and Use Committee of Chongqing University, and were in accordance with the National and Institutional Guidelines for Animal Care. Ten-week-old male C57BL/6 mice were provided by the Experimental animal Center of Chongqing Medical University. NLRP3eviously . AdditioPulmonary edema was evaluated by lung wet to dry wet ratio as described previously . BrieflyMice were treated with heparin and anesthetizing. A small incision in the trachea was made with the tips of forceps. Then 3 mL cold PBS was injected into lungs through the incision, the fluid was sucked out after massaging the chest. Repeating this perfusion procedure for three times and collected about 1.5 mL fluid. The collected fluid was subsequently centrifuged at 1000 rpm for 10 min at 4℃. The protein content in BALF was evaluated with a BCA assay kit.The levels of TNF-α and IL-1β in BALF as well as medium were detected with TNF-α and IL-1β Elisa kit  according to the manufacturer’s instructions. The concentration of S100A9 in BALF and lung homogenates was measured with a mouse S100A9 ELISA kit  according to the manufacturer's instruction.Lung homogenates was used for evaluating the MPO activity and LDH content with corresponding assay kit  according to the manufacturer's instruction.g for 15 min at 4 ℃, the supernatant was collected. Then the lysates were incubated with Ac-DEVD-pNA at 37℃ for 100 min before detected.The caspase-3 activity was measured with Caspase 3 Activity Assay Kit  according to the manufacturer's instruction. The lung tissues were homogenized in lysis buffer. After centrifugation at 16000Mice were treated with heparin and anesthetized with 2% isoflurane. Subsequently, the left lung was harvested and fixed in 4% paraformaldehyde. Tissues were embedded in paraffin and cut into 7-μm-thick sections for hematoxylin and eosin (H&E) staining. The tissue pathology was assessed based on the inflammatory cell infiltration, edema degree and the thickness of alveolar septum in at least five random fields under a light microscope  [Total RNA form lung tissue was extracted using Trizol regent , 1000 ng RNA was reverse transcribed with Superscript. Subsequently, qRT-PCR was performed with SYBR green reagent on Bio-rad real time PCR system. Primers used for measuring gene expression were shown below: IL-1β, TNF-α  and β-actin .The human lung epithelia cells line, BEAS-2B, was purchased from China Cell Line Bank. The cells were maintained in DMEM with 10% fetal bovine serum , 0.022% sodium pyruvate and 0.26% sodium bicarbonate. To overexpress NLRP3, cells were transfected with plasmids encoding NLRP3  by Lipofectamine 3000  for 24 h. Then the cells were treated with 100 ng/ml LPS for 8 h followed by 5 mM ATP for 1 h, with or without 10 μM ARB starting at 24 h after transfection.Heart tissues or cells were lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology), and the protein extracts were then centrifugated at 12,100 rpm for 15 min. Subsequently, the protein concentration was measured with a BCA Protein Assay. A total of 30 μg protein extracts were separated by SDS-PAGE and were then electrotransferred onto polyvinylidene fluoride membranes. Following blocking with 5% non-fat milk for 1 h at room temperature, the membranes were first probed with primary antibodies overnight at 4 °C, and then with the corresponding secondary antibodies. The protein blots were visualized using an ECL imaging system .Lung tissues were lysed with NP-40  followed by centrifugation at 12,000 RPM for 15 min at 4 ℃ and the supernatant was collected. protein concentration were measured by BCA protein assay kit . Then 200 μg lysates were incubated with Protein A magnetic Beads  with a tube rotator at 100 revolutions per minute for 2 h at 4 °C. After the beads were discarded by magnetic force, the antibody of S100A9  was added to the precleared lysate at 4 °C for overnight. After aspirating the supernatant, the complex were washed with NP-40 buffer with 500 nM NaCl for 5 times and then resuspended in sample buffer, followed by incubation at 98 °C for 10 min to release the proteins form beads. Then the protein was used for western blot to detect the interaction of S100A9 and TLR4.All values are expressed as the mean ± standard error of the mean (SEM). Data were analyzed with one-way or two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. Survival curves were constructed using the Kaplan–Meier survival curve and analyzed using a log-rank test. P < 0.05 was considered to indicate a statistically significant difference.Firstly, the degree of lung injury and the expression of S100A9 in the lung tissues of LPS-treated mice was measured at 4, 12 and 24 h following treatment with LPS. The results showed that LPS significantly induced lung injury starting at 4 h (Fig. To evaluate whether S100A9 serves a pathogenic role in LPS-induced lung injury, the effects of S100A9 blockade on lung injury were determined. Therefore, mice were co-treated with 20 or 30 mg/kg of the specific S100A9 blocker, ABR, and LPS. Following treatment for 24 h, the lung tissues and BALF were collected. Compared with the control group, treatment with LPS resulted in significant lung injury, evidenced by increased inflammatory cell infiltration (Fig. S100A9 is mainly produced and sorted in neutrophils and macrophages, and S100A9 is considered as an important inflammatory alarmin for triggering immune responses . SubsequIt has been reported that another member of the S100 family, S100A12, promotes sepsis-induced ARDS via activating the NLRP3 pathway . In lineTo investigate whether S100A9 blockade could act via suppressing the NLRP3 pathway, the lung epithelial cells, BEAS-2B, were transfected with NLRP3 overexpression plasmids. The transfection efficiency was evaluated by western blot analysis Fig. a, b. Sub−/− mice (Fig. −/− mice (Fig. To further verify whether S100A9 blockade could act via blocking the NLRP3 pathway in vivo, the effects of S100A9 inhibition were determined in wild-type and NLRP3 null mice. The results showed that genetic silencing of NLRP3 could not affect the interaction of S100A9 with TLR4 in the lung tissues of LPS-treated mice, suggesting that NLRP3 was not the upstream molecule of S100A9 Fig. a. In addice Fig. b–d. Moreice Fig. e–g. Althice Fig. h, i. Thi2+ binding protein, which is constitutively stored in neutrophils and monocytes as a Ca2+ sensor [LPS-induced lung injury is characterized by excessive inflammation and accumulation of inflammatory cells, especially neutrophils and macrophages, in the lungs . Neutrop+ sensor . Upon in+ sensor . It has + sensor , 31.Herein, LPS-induced lung injury resulted in increased S100A9 levels in the lung tissues and BALF. To investigate the functional roles of S100A9 in LPS-induced lung injury, mice were treated with a S100A9 inhibitor, at 0 and 24 h following LPS administration. The results showed that the pharmacological inhibition of S100A9 led to marked reduction in tissue injury and cell apoptosis. Consistent with the previous finding, the inflammatory responses were also significantly mitigated by blockade of S100A9. These findings supported the hypothesis that S100A9 could be considered as an important inflammatory mediator contributing to the progression of LPS-induced lung injury, and that inhibiting S100A9 could protect against LPS-induced lung injury in mice. However, a previous study reported that S100A9 attenuated acute lung injury, which was inconsistent with the results of the current study. This discrepancy could be due to the differences in the LPS stimulation process  or becau−/− mice. In addition, blockade of S100A9 could not provide additional protective effects in NLRP3−/− mice. In vitro, NLRP3 overexpression blunted the effects of S100A9 inhibition on attenuating the production of proinflammatory cytokines, which further supported that the effects of S100A9 inhibition on LPS-induced lung injury could be associated with the suppression of the NLRP3 inflammasome activation.It has been demonstrated that excessive S100A9 accelerates the production of cytokines by macrophages and neutrophils, thus aggravating the inflammatory responses , 35. TheIt has been also reported that NLRP3 may cause apoptosis . TherefoOne limitation of the present study is that a pharmacological inhibitor was used to block S100A9, thereby, the non-target effects of the chemical inhibitor could not be excluded. Therefore, further verifying the results of the current study using a genetically modified mouse model is of great importance.In conclusion, the present study suggested that S100A9 could serve a key role in LPS-induced lung injury by triggering excessive inflammatory responses, partially via the NLRP3 pathway. Furthermore, S100A9 blockade alleviated lung injury in mice, thus providing a potential novel therapeutic target against acute lung injury.


Clonorchis sinensis, and investigated its protective effects against C. sinensis challenge.Clonorchiasis is endemic in East and Southeast Asian countries. For a preventive strategy against infectious diseases, vaccination is the most effective. Here, we evaluated the molecular characteristics and immune responses of CsAg17 protein from C. sinensis transcriptome bank. Recombinant CsAg17 B-cell epitope protein and cDNA vaccines were produced and their immune responses were evaluated in FVB mice. The proportional changes of CD3+/CD4+ and CD3+/CD8+ T cells were detected by flow cytometry, and immune effectors were measured by ELISA.A cDNA clone encoding CsAg17 protein and containing a secretory signal peptide at the N-terminus was retrieved from the CsAg17 mRNA was transcribed at a higher level in C. sinensis adults than in metacercariae. The CsAg17 protein was distributed in the sperms, oral and ventral suckers, and mesenchymal tissues of C. sinensis adults. In mice challenged with C. sinensis metacercariae, vaccination with CsAg17 protein and cDNA resulted in a reduction to 64% and 69% in worm burden, respectively. Both CsAg17 protein and cDNA vaccines increased the proportion of CD3+/CD4+ and CD3+/CD8+ T cells and stimulated the production of Th1 type cytokines such as interleukin (IL)-2, IL-12, and interferon-γ, while maintaining minimum levels of Th2 cytokines. The levels of IgG specific to CsAg17 protein steeply increased in the two vaccinated groups from 2 weeks after immunization. The liver tissue retained good morphology in the mice vaccinated with CsAg17 protein or cDNA, whereas severe inflammation and large serous cysts were observed in the liver of the unvaccinated mice.The via cellular and humoral immune responses. Thus, they may serve as good vaccine candidates against C. sinensis infections.Vaccination with CsAg17 protein and cDNA reduced the pathological changes in the bile duct and liver, and ameliorated the worm burden  Clonorchis sinensis ; Sigma-Aldrich, St. Louis, MO, USA) was added to the plate and incubated at 37 °C for 1 h. The samples were treated with the substrate 6 cells) were isolated from 3 mice from each group before and after challenge infection, washed twice with PBS (pH 7.4), and resuspended in PBS containing 10% fetal bovine serum (FBS). The splenocytes were incubated with anti-mouse CD16/CD32 antibody and monoclonal antibody of fluorescein isothiocyanate (FITC)-labeled CD4, phycoerythrin (PE)-labeled CD8, and allophycocyanin (APC)-labeled CD3  in a final reaction volume of 20 μl for 30 min on ice in the dark. The cells were washed once with PBS and fixed with 500 μl of 5% formaldehyde. After washing with PBS twice, the cells were resuspended in 1 ml PBS and loaded on a flow cytometry machine . Cells were selected using the CD3 signal as gate R1 and the proportion of CD4+ or CD8+ subpopulation in CD3+ T cells was analyzed. Data were analyzed using BD CellQuestTM Pro software.The splenocytes . Concanavalin A  was used as a positive control in all assays. All plates were incubated for 48 h at 37 °C in a 5% CO2 humidified atmosphere. Supernatant from the splenocyte culture was collected and incubated at −20 °C. Interleukin (IL)-2, IL-4, IL-10, IL-12 and interferon-γ (IFN-γ) in the supernatant were measured using sandwich ELISA kits (eBioscience). The procedure was as follows: Corning Costar 9018 ELISA plates  were coated with 100 μl/well of IL-2, IL-4, IL-10, IL-12 or IFN-γ capture antibody in 1× coating buffer and incubated overnight at 4 °C. The medium was aspirated, and the plates were washed thrice, followed by blocking with 1× ELISA diluent (200 μl/well) and incubation at 25 °C for 1 h. After washing, 2-fold serial dilutions of standard IL-2, IL-4, IL-10, IL-12 and IFN-γ or splenocyte culture supernatant (100 μl/well) were added to the respective wells and the plates were incubated at 25 °C for 2 h. After washing 3–5 times, detection antibody (100 μl/well) was added to each well and the plate was incubated at 25 °C for 1 h. After washing 3–5 times, the plate was incubated with avidin-horseradish peroxidase  at 25 °C for 30 min. The plate was washed 5–7 times and incubated with 1× TMB solution (100 μl/well) at 25 °C for 15 min, followed by the addition of 50 μl of 2 N H2SO4. Optical density was measured at a wavelength of 450 nm using a microplate reader . All assays were performed in triplicate.Splenocytes from each group were isolated and seeded at a density of 5 × 10Four weeks after infection, mice were sacrificed, and their livers were excised and observed by naked eye. The livers were fixed in 10% paraformaldehyde, embedded in paraffin, and sectioned into ribbons. The ribbons were deparaffinized, rehydrated, and placed on slides. The slides were stained with a nuclear dye (hematoxylin) and rinsed, followed by counterstaining with eosin. The slides were sequentially rinsed in water, alcohol, and xylene and covered with coverslips. Pathological changes were observed under a light microscope and photomicrographs were obtained.P < 0.05 were considered statistically significant. All values represent the mean ± standard error (SE) of the mean of more than three independent assays.Data were analyzed using the Student’s t-test and values of C. sinensis and a good vaccine candidate. The CsAg17 cDNA sequence was deposited in the GenBank database under the accession number MN381946.CsAg17 cDNA was 420 bp in length and predicted to encode a polypeptide of 139 amino acid (aa) residues, with an estimated molecular weight of 15.1 kDa. This polypeptide had five helical regions, one of which was the transmembrane domain. A putative secretory signal peptide of 22 aa was predicted with a cleavage site at the N-terminus. The tertiary structure of CsAg17 protein was predicted to have five α-helices and loops , and remained in the soluble form. As a fusion protein to 6× His tag, CsAg17 was purified using Ni-NTA column under native conditions. The protein appeared as a single band on sodium dodecyl sulfate polyacrylamide gel and had an expected molecular weight of 10 kDa  values of target and reference genes using the 2−ΔΔCq method [CsAg17 mRNA was 2.5-fold higher in the adults than in the metacercariae . Lane 1: uninduced E. coli lysate; Lane 2: total lysate of induced E. coli; Lane 3: soluble fraction of induced E. coli lysate; Lane 4: pass-through fraction; Lane 5; wash-off; Lanes 6, 7: first and second eluates; Lane M: protein molecular weight marker.Additional file 2: Figure S2. Immunoblotting of CsAg17 proteins using mouse anti-CsAg17 B cell epitope immune serum. Lane 1: soluble extract of C. sinensis adult; Lane 2: recombinant CsAg17 B cell epitope and normal mouse serum; Lane 3: insoluble recombinant full length CsAg17; Lane 4: recombinant CsAg17 B cell epitope; Lane M: protein molecular weight marker.Additional file 3: Figure S3. Localization of CsAg17 protein in C. sinensis adults. Tissues were treated with mouse anti-CsAg17 partial protein immune serum (a–e), or normal mouse serum (f–j). Abbreviations: SR, seminal receptacle; VS, ventral sucker; Ms, mesenchymal tissue; Egg, intra-uterine egg; In, intestine.Additional file 4: Figure S4. Transcription level of CsAg17 mRNA during developmental stages. RT-qPCR was performed with the mRNAs obtained from C. sinensis metacercariae and adults. Relative transcription level of CsAg17 mRNA is shown. ***P < 0.001 compared to metacercariae.


To the editor:mACE2 (membrane-bound angiotensin-converting enzyme 2) is central when developing severe COVID-19 (coronavirus disease 2019), because SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) attaches to the active surface domain of mACE2 when entering the host cell , a proceSevere COVID-19 is more common in adults than in children and in men than women . This mahttp://www.olink.com). Seven outlier observations of sACE2 that deviated more than 3 SD from the gender-specific mean were removed from the analyses. Characteristics of subjects included in the analyses of the present study are presented in Table p < 0.05, and analyses were performed using the SPSS statistical package .We analyzed sACE2 in serum collected at mean ages (SD) 9.9 0.6), 11.7 (0.6), 14.8 (0.8), 18.8 (0.3), and 23.5 (0.7) years in individuals in the pediatric osteoporosis prevention (POP) study, a prospective study that investigates the effects of physical activity on musculoskeletal development through growth  [, 11.7 . We suggest that further studies evaluate if high sACE2 is a risk factor for severe COVID-19 and to what extent sACE2 is related to increased ADAM-17 activity and mACE2 shedding.


Wisteria floribunda agglutinin-positive mac-2 binding protein (WFA±M2BP) ≥ 1.85 and α-fetoprotein (AFP) ≥ 6.0 ng/mL were independent factors of HCC development. However, at 1 year after SVR24, WFA±M2BP ≥ 1.85 was associated with subsequent HCC development  but not AFP. Among patients with WFA±M2BP ≥ 1.85 at SVR24, 42% had WFA±M2BP < 1.85 at 1 year after SVR24 (WFA±M2BP declined group). Subsequent HCC development was significantly lower in the declined group than in the non-declined group . In conclusion, WFA±M2BP but not AFP could identify high and no-risk cases of HCC at 1 year after SVR. Therefore, it was useful as a real-time monitoring tool to identify the candidates for continuous surveillance for HCC.Identification of high-risk patients for hepatocellular carcinoma (HCC) after sustained virological responses (SVR) is necessary to define candidates for long-term surveillance. In this study, we examined whether serum markers after 1 year of SVR could predict subsequent HCC development. Total 734 chronic hepatitis C patients without a history of HCC who achieved SVR with direct-acting antivirals were included. The regular surveillance for HCC started from 24 weeks after the end of treatment (SVR24). Factors at SVR24 and 1 year after SVR24 were analyzed for predicting HCC development. During the mean observation period of 19.7 ± 10 months, 24 patients developed HCC. At SVR24,  Direct-acting antivirals (DAAs) treatment for chronic hepatitis C has enabled sustained virological responses (SVR) in several patients in recent years ,3,4,5,6.Wisteria floribunda agglutinin-positive mac-2 binding protein (WFA±M2BP) is a novel serum fibrosis marker [±M2BP at SVR is reported to be associated with HCC development [±M2BP and AFP can identify patients at high risk of HCC development at the time of SVR, it is difficult to continue screening for all cases judged high risk at the time of SVR. Liver fibrosis changes after the achievement of SVR differ from case to case [Non-invasive fibrosis markers and α-fetoprotein (AFP) levels at 12 or 24 weeks after the end of treatment (SVR12 or 24) are reportedly associated with subsequent HCC development ,11,12,13s marker ,15, and elopment ,17. Alth to case , and sub to case ,20. Ther±M2BP and AFP at 1 year after SVR are associated with HCC development. Further, it is unclear whether changes in the fibrosis markers alter the risk of carcinogenesis. Therefore, we examined whether WFA±M2BP and AFP after 1 year of achieving SVR are associated with HCC development and whether these changes could be used to evaluate the risk of HCC.Serum markers can be simply and repeatedly measured; therefore, it is considered possible to reevaluate the risk of HCC over time and further narrow down the high-risk cases. However, to our knowledge, no report has examined whether WFAThe clinical characteristics and laboratory data of patients are described at the time point of SVR24 and one year after SVR24 in ±M2BP and fibrosis stage was examined. Median value of WFA±M2BP in F1, F2, F3, and F4 was 0.75, 1.16, 2.06, and 3.01, respectively, and WFA±M2BP increased as fibrosis stage increased (p < 0.001).Association between WFA±M2BP and AFP at SVR24 were analyzed for predicting HCC development. ROC analysis was used to select WFA±M2BP of 1.85 cut off index (COI) as the optimal cutoff value for predicting HCC development within 1 year. WFA±M2BP of <1.85/≥1.85 were defined as low/high risk and low/high-risk patients were 567 (77.2%), and 167 (22.8%), respectively. The AFP level of 6.0 ng/mL was selected as the cutoff value and AFP of <6.0/≥6.0 ng/mL was defined as low/high risk of AFP. The 1-, 2-, and 3-year rate of HCC development in patients with low/high risk of WFA±M2BP were 1.2%/1.5%/1.5%, and 8.1%/13.1%/14.6%, respectively. The cumulative rate of HCC development was higher in patients with high risk than those with low risk . Serum WFA±M2BP and AFP at 1 year after SVR24 (78 weeks post-treatment) were analyzed for predicting HCC development thereafter. Using the same cutoff values (WFA±M2BP of 1.85 COI and AFP level of 6.0 ng/mL), the cumulative incidence of HCC development was examined. Seven patients developed HCC after 1 year of SVR. The 1- and 2-year rate of HCC development, starting from the time point of 78 weeks post-treatment, in patients with low/high risk of WFA±M2BP at 1 year after SVR24 were 0.3%/0.3%, and 8.6%/11.3%, respectively . Similarly, the AFP level at SVR24 was 3.99 ± 3.4 ng/mL and decreased significantly to 3.51 ± 2.2 ng/mL 1 year after SVR24 (p < 0.001).Time-course changes in WFA±M2BP (≥1.85 COI) at SVR24, WFA±M2BP decreased to < 1.85 COI (low risk) at 1 year after SVR24 in 42 patients  (WFA±M2BP declined group) and remained ≥ 1.85 COI in 60 patients (60%) (WFA±M2BP non-declined group). Among patients with a high risk of AFP (≥6.0 ng/mL) at SVR24, AFP decreased to < 6.0 ng/mL at 1 year after SVR24 in 30% (20/67) patients (AFP declined group) and remained ≥ 6.0 ng/mL in 70% of the patients (AFP non-declined group). The 1- and 2-year rates of HCC development, starting from the time point of 78 weeks post-treatment, were 0% and 0%, respectively, in the WFA±M2BP declined group, while these rates were 9.4% and 12.4%, respectively, in the WFA±M2BP non-declined group. The cumulative incidence of HCC development was significantly higher in patients in the WFA±M2BP non-declined group . However, at 1 year after SVR24, 0% of the patients with AFP ≥ 6.0 ng/m and 2.1% of those with AFP < 6.0 ng/m had LR3/4 nodules, with no significant difference.The association between serum AFP levels and the presence of LR3/4 nodule was analyzed at the time point of SVR24 and 1 year after SVR24. At SVR24, 2.1% (13/623) of patients with AFP at SVR24 < 6.0 ng/m had LR3/4 nodules and 7.2% (8/111) of patients with AFP ≥ 6.0 ng/m had LR3/4 nodules, and presence of LR3/4 nodules was significantly higher in patients with AFP ≥ 6.0 ng/mg , albumin, AST (every 30 IU/L), ALT (every 30 IU/L), platelet counts, WFA±M2BP ≥ 1.85 COI : 3.91–22.7, p < 0.001, p < 0.001), and presence of LR3/4 nodules  were associated with HCC development in the univariate analysis. By using these factors, multivariate analysis revealed that WFA±M2BP ≥ 1.85 COI , AFP ≥ 6.0 ng/mL , and the presence of LR3/4 nodules  were independently associated with HCC development.Factors at SVR24, including those other than WFA±M2BP and AFP, were analyzed for their association with HCC development. Similar to SVR24, WFA±M2BP ≥ 1.85 COI , albumin, platelet counts, and presence of LR3/4 nodules  at 1 year after SVR24 were associated with HCC development in the univariate analysis. However, AFP ≥ 6.0 ng/mL at 1 year after SVR24 was not associated with subsequent HCC development . Multivariate analysis revealed that WFA±M2BP ≥ 1.85 COI  and presence of LR3/4 nodules  were independent factors at 1 year after SVR24 for the prediction of HCC development thereafter.Similarly, factors at 1 year after SVR24, including those other than WFA±M2BP was useful for predicting HCC development at 1 year of SVR; however, AFP was not useful. In addition, even if WFA±M2BP was high (≥ 1.85 COI) at SVR, the risk of HCC decreases in patients in whom WFA±M2BP subsequently declines (<1.85 COI). However, AFP was useful for predicting HCC at the time of SVR, but not after one year. WFA±M2BP could be easily measured and helped identify high cases of HCC development at any time after SVR; therefore, it was useful as a real-time monitor of HCC development risk in the long-term follow-up after SVR.The present study revealed that WFA±M2BP after 1 year of SVR is associated with HCC development risk. The association between WFA±M2BP at SVR and HCC risk has been reported by several studies, including our report [±M2BP and HCC development risk after long-term follow-up. Early detection of HCC development after DAA treatment requires long-term follow-up. Therefore, not only the prediction of HCC development at SVR time point but also the prediction of HCC development at each follow-up time point is important. We found that WFA±M2BP ≥ 1.85 COI at 1 year of SVR, as well as SVR24, were factors for HCC development.One of the novel findings of this study was that it confirmed that a high level of WFAr report ,17. Sinc±M2BP after SVR, WFA±M2BP generally declines over time. Patients with WFA±M2BP ≥ 1.85 COI at SVR were at high-risk for subsequent HCC development; however, those with decreased WFA±M2BP after 1 year had a reduced risk of HCC development. However, in some cases, WFA±M2BP was still high, and in such cases, the risk of HCC development remained high. The advantage of WFA±M2BP is that the change in the HCC risk can be identified by observing the time-dependent changes in WFA±M2BP.Considering the changes in WFA±M2BP is useful as a real-time monitor for the prediction of HCC development in that WFA±M2BP can assess the risk of HCC at any time point and assess changes in HCC risk by observing changes over time; further, clinically important information can be obtained noninvasively and conveniently.Although long-term follow-up is necessary to identify patients with HCC development after SVR, it is difficult to screen all patients frequently. However, the lack of screening after SVR is a known risk factor for the development of advanced HCC . Therefo±M2BP, rather than AFP, was useful for HCC monitoring during long-term follow-up after SVR.Another novel finding of this study was that AFP after 1 year of SVR was not useful for predicting HCC development. It has been widely reported that AFP levels at SVR are associated with HCC risk ,11,12,17±M2BP over time, it is necessary to verify whether WFA±M2BP of 2 years after SVR24 and later is useful for prediction of HCC development, which is a future study subject. Moreover, there were few HCC cases; therefore, future long-term studies on a larger cohort are needed to examine the usefulness of WFA±M2BP over time. Several fibrosis markers have been confirmed as a predictive marker of HCC development after SVR [±M2BP and other markers in future studies. There are certain limitations to this study. At present, few cases have been followed up for 2 years. To verify the usefulness of WFAfter SVR ,25. The ±M2BP but not AFP was useful as a real-time monitor of HCC prediction in long-term follow-up after SVR.In conclusion, WFABetween October 2014 and March 2018, 871 patients received DAAs at the Musashino Red Cross Hospital for the treatment of chronic hepatitis C and achieved SVR. Of these, 734 who met the following criteria were enrolled in this study: (1) those who were followed up for 6 months or more after SVR24, (2) had no history of HCC development, and (3) had no co-infection with hepatitis B virus or human immunodeficiency virus. The observation period began at the time of SVR24, and HCC development after SVR was followed up. Written informed consent was obtained from each patient. The study protocol conformed to the ethical guidelines of the Declaration of Helsinki and was approved by the institutional ethics review committee .Age and sex of the patients were recorded at SVR24. Fasting blood counts and biochemical tests were also conducted on SVR24 and 1 year after SVR24 using standard methods.Ultrasonography and blood tests, including tests for tumor markers, were performed every 3–6 months for HCC surveillance. When tumor marker levels rose abnormally and/or abdominal ultrasonography suggested a lesion suspicious of HCC, contrast-enhanced computed tomography, magnetic resonance imaging, or angiography was performed. HCC was diagnosed for tumors displaying vascular enhancement at the early phase and washout at the later phase as per the guidelines of the American Association for the Study of Liver Diseases, and the Japan Society of Hepatology ,27. TumoOf all patients enrolled in the study, liver biopsy was performed in 257 patients 1 month prior to the initiation of DAA treatment until the treatment. Liver biopsy specimens were obtained laparoscopically using 13G needles or by percutaneous ultrasound-guided liver biopsy using 15G needles. Specimens were fixed, paraffin-embedded, and stained using hematoxylin–eosin and Masson’s trichrome. A minimum of a 15-mm biopsy sample was required for diagnosis. All liver biopsy samples were independently evaluated by two senior pathologists who were blinded to the clinical data. Fibrosis staging was assessed according to the METAVIR score: F0, no fibrosis; F1, portal fibrosis without septa; F2, portal fibrosis with few septa; F3, numerous septa without cirrhosis; and F4, cirrhosis.±M2BP and AFP for predicting HCC development. Statistical significance was defined as a p-value < 0.05. Cumulative incidences of HCC development were calculated using the Kaplan–Meier method. The factors associated with HCC development were analyzed using the Cox-proportional hazard model. Correlated factors with a p-value < 0.05 in the univariate analysis were used for further multivariate analysis. Backward stepwise selection method was used for multivariate analyses. Association between WFA±M2BP and fibrosis stage was analyzed using Spearman’s rank correlation test. Statistical analyses were performed using EZR  [Receiver operating characteristic (ROC) curves and the Youden index were used to determine the optimal cutoff value of WFA, Japan)  and a gr