1,25(OH)2D3 Promotes Macrophage Efferocytosis Partly by Upregulating ASAP2 Transcription via the VDR-Bound Enhancer Region and ASAP2 May Affect Antiviral Immunity

The active form of vitamin D3, i.e., 1,25(OH)2D3, exerts an anti-inflammatory effect on the immune system, especially macrophage-mediated innate immunity. In a previous study, we identified 1,25(OH)2D3-responsive and vitamin D receptor (VDR)-bound super-enhancer regions in THP-1 cells. Herein, we examined the transcriptional regulation of ArfGAP with SH3 Domain, Ankyrin Repeat and PH Domain 2 (ASAP2) (encoding a GTPase-activating protein) by 1,25(OH)2D3 through the top-ranked VDR-bound super-enhancer region in the first intron of ASAP2 and potential functions of ASAP2 in macrophages. First, we validated the upregulation of ASAP2 by 1,25(OH)2D3 in both THP-1 cells and macrophages. Subsequently, we identified three regulatory regions (i.e., the core, 1,25(OH)2D3-responsive, and inhibitory regions) in the VDR bound-enhancer of ASAP2. ASAP2 promoted RAC1-activity and macrophage efferocytosis in vitro. Next, we assessed the functions of ASAP2 by mass spectrometry and RNA sequencing analyses. ASAP2 upregulated the expressions of antiviral-associated genes and interacted with SAM and HD domain-containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1). In vivo, vitamin D reduced the number of apoptotic cells in experimental autoimmune encephalomyelitis (EAE) and promoted macrophage efferocytosis in peritonitis without changing the mRNA level of ASAP2. Thus, we could better understand the regulatory mechanism underlying ASAP2 transcription and the function of ASAP2, which may serve as a potential treatment target against inflammatory diseases and virus infections.


Introduction
Vitamin D plays an important role in anti-inflammation partly by regulating the innate immune system, especially macrophages. For example, vitamin D can inhibit the production of pro-inflammatory cytokines in monocytes and macrophages by regulating mitogenactivated protein kinase phosphatase 1 [1]. Furthermore, it can downregulate IL-8 expression in hyperinflammatory macrophages, thus serving as a potential anti-inflammatory treatment strategy for chronic inflammatory lung disease [2]. 1,25(OH) 2 D 3 , the active form of vitamin D 3 , also promotes anti-infectious innate immune responses, such as upregulation of the expressions of antimicrobial peptides β-defensin 2 and cathelicidin antimicrobial peptide [3]. Vitamin D receptor (VDR), the only cognate receptor of the active form of vitamin D 3 (i.e., 1,25(OH) 2 D 3 ), as a transcription factor (TF), can bind multiple genomic regions, thus interacting with multiple other TFs and coregulators for transcriptional regulation [4].
Super-enhancers (SEs) are dense clusters of enhancers identified by chromatin accessibility assays (Formaldehyde-Assisted Isolation of Regulatory Elements-sequencing (FAIRE-seq) identified regions or DNaseI hypersensitive sites), pioneer/master TFs (e.g., PU.1 for monocytes and RORγt for Th17 cells), and pervasive factors in the transcription machinery (e.g., p300, MED1, BRD4, and RNA polymerase II). These can strongly promote gene transcription and predict key genes having cell-specific and stimulation-responsive functions [5,6]. The regulation of 1,25(OH) 2 D 3 on its target genes is strictly dependent on spatio-temporal VDR binding sites (including 1,25(OH) 2 D 3 -driven super-enhancers) in the context of chromatin (e.g., cellular, temporal, and species specificity) [7][8][9]. In our previous study, we identified three genes (i.e., DENN Domain Containing 6B (DENND6B), Ubiquitin Specific Peptidase 2 (USP2), and ArfGAP with SH3 Domain, Ankyrin Repeat and PH Domain 2 (ASAP2)) showing high expressional levels and top-ranked type I VDR super-enhancers (i.e., the VDR super-enhancer (VSE) regions without pre-occupied VDR but transforming to VSE after 1,25(OH) 2 D 3 stimulation) in THP-1 cells [6]. Although the intensity of ASAP2 VSE is high enough for identification as a super-enhancer in a range of 12.5 kb using the rank ordering of super-enhancers (ROSE) algorithm, the full length of ASAP2 VSE is only 1146 bp. Therefore, we speculated it as a classic VDR-bound enhancer exerting a strong effect on gene transcription.
ASAP2 protein is an ADP-ribosylation factor GTPase-activating protein (ARFGAP) that can activate downstream GTPase ARF6 and then RAC1, thus regulating vesicular transport and FcγR-mediated phagocytosis [10][11][12]. ASAP2 protein has multiple functional domains including the ARFGAP domain exhibiting GTPase-activating protein (GAP) activity. Owing to the persistent GTPase activities (i.e., ARF6 and RAC1), cycling between two forms-GTPase-GTP and GTPase-GDP-is necessitated. Both overexpression and mutation of ASAP2 lead to the inhibition of the F-actin assembly at the phagocytosis cup [10,13,14]. Selenoprotein K-dependent palmitoylation of ASAP2 mediates the dissociation of ASAP2 from the phagocytic cup, crucial to the efficiency of FcγR-mediated phagocytosis [15]. Because actin assembly and cytoskeletal remodeling occur in both phagocytosis and efferocytosis, and the latter plays an important role in anti-inflammatory effects by preventing secondary necrosis and resultant inflammation [16], we hypothesized that 1,25(OH) 2 D 3upregulated ASAP2 expression could promote efferocytosis in THP-1 derived macrophages and attenuate tissue inflammation.
Therefore, herein, we examined the transcriptional regulatory mechanism underlying 1,25(OH) 2 D 3 action on ASAP2 via its VDR-bound enhancer region and confirmed the enhancing effect of 1,25(OH) 2 D 3 and ASAP2 on efferocytosis both in vitro and in vivo. Furthermore, we assessed other functions of ASAP2 by mass spectrometry and RNAsequencing (RNA-seq) analyses. We provided evidence for the association between 1,25(OH) 2 D 3 and efferocytosis in an inflammatory disease model, which requires further validation in the future.
Peripheral blood mononuclear cells (PBMCs) were obtained from healthy adult donor buffy coats using Ficoll-Paque Plus (#17144002, Cytiva, Uppsala, Sweden) by gradient centrifugation following the manufacturer's protocol. The experiment was approved by the Biomedical Ethics Committee of Anhui Medical University (No. 20190324). PBMCs were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% 100× penicillin and streptomycin overnight. The next day, the medium was removed and replaced with fresh media. Subsequently, the cells were treated with fresh medium containing 20 ng/mL human M-CSF (#C417, Novoprotein, Suzhou, China) for 7 days. Macrophages were incubated in a fresh medium with or without 100 nM 1,25(OH) 2 D 3 for 48 h.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted using the TRIzol reagent (#YY101, Epizyme, Shanghai, China) following the manufacturer's protocols and reverse-transcribed into cDNA using Hifair ® III 1 st Strand cDNA Synthesis SuperMix (#11141ES60, YEASEN, Shanghai, China) for qRT-PCR. qRT-PCR was performed using Hieff ® qPCR SYBR Green Master Mix (#11201ES50, YEASEN, Shanghai, China) on the Lightcycler 96 real-time PCR system (Roche, Switzerland). The primer sequences used in this study are listed in Table S1. Relative gene expression was determined by the 2 −∆∆Ct method. The expressional levels of genes were normalized to the expression of GAPDH, i.e., a "housekeeping" gene, as an endogenous reference. The primer sequences used in this study are shown in Supplementary File Table S1.

Stable Knockdown of ASAP2
The shRNAs were chemically synthesized by Genechem, China. Oligos for shRNA targeting ASAP2 (target sequence: shNC TTCTCCGAACGTGTCACGT; shASAP2-1 ccGGT-GTCATTTGTGCACTTT, shASAP2-2 ccTGGATAAACAGACAGGGAA, and shASAP2-3 gcCTCAAACCTTCCATTGAAA) were inserted into the GV493 vector. Lentiviruses were prepared from 293T cells transfected with GV493 vector and packaging plasmid mix (Helper 1.0 and Helper 2.0, Genechem, Shanghai, China). THP-1 cells were seeded in a 12-well plate, and the lentiviral construct (LV-ASAP2-RNAi) was transfected. After culturing at 37 • C for 12-16 h, a fresh complete medium was added to the culture. Cells were selected in 2 µg/mL puromycin, 48 h after infection, for another 48 h. Cells were then cultured in fresh medium for the next experiment or RNA-seq analysis, whereby THP-1 cells were differentiated into macrophages by adding 50 ng/mL PMA for 48 h. Our RNA-seq data are in the GEO database with accession no. GSE271201.

Chromatin Immunoprecipitation (ChIP) Assay
The ChIP assay for VDR was performed using an Enzymatic Chromatin IP Kit (#9005, CST, MA, USA). The lysates of THP-1-derived macrophages were sheared by sonication and incubated with micrococcal nuclease to generate DNA fragments. Antibodies against VDR (#ab109234, ABCAM, Cambridge, UK) and normal rabbit IgG (#2729, CST, MA, USA) were used for immunoprecipitation. Normal rabbit IgG was used as the negative control. The ChIP products were quantified by PCR using specific primers (VSE: Forward Primer: GCCCTTCAGAAGATTCTGAAA, Reverse primer: CACACTAGCATTTGAAGTTCAC) followed by gel electrophoresis.

Pulldown Assay for Testing RAC1 Activity
THP-1 monocytes were plated in a 10 cm Petri dish at the density of 5 × 10 6 cells/mL and differentiated into macrophages after stimulation with 50 ng/mL PMA. After coculturing with apoptotic Jurkat cells for 2 h, lysates of THP-1-derived macrophages were prepared using the Rac1 Activation Magnetic Beads Pulldown Assay kit (#17-10394, Merck Millipore, Darmstadt, German). Glutathione S transferase (GST) fused to the p21 binding domain of p21-activated kinase PAK1 (GST-PAK1 PDB), and glutathione magnetic beads in the kit were used for lysate incubation at 4 • C for pulling down RAC1-GTP after washing thrice with magnesium lysis buffer. The levels of RAC1-GTP in the pulldowns and the total RAC1 in the whole lysates were compared by Western blot analysis.

Histological Analysis and Immunofluorescence Staining
For EAE, the spinal cords were flushed with PBS from the spinal columns of sacrificed mice and fixed overnight in 4% paraformaldehyde (PFA, #BL539A, Biosharp, Hefei, China). Tissue sections were stained with hematoxylin and eosin (H&E) or Luxol fast blue (LFB) for evaluating inflammation and demyelination, respectively. Slides were visualized by light microscopy using a Panoramic tissue cell quantitative analyzer (TG, Austria). For assessment of apoptosis/efferocytosis in the spinal cord, detection of the apoptotic cells in tissues was performed using a Fluorescein (FITC) TUNEL Cell Apoptosis Detection Kit (#G1501, Service, Wuhan, China) following the manufacturer's protocol. The nuclei were counterstained with DAPI (#MX4209, Maokang, Shanghai, China) for 5 min. Slides were visualized using a Nikon fluorescent microscope (Nikon, Japan).

Statistical Analyses
GraphPadPrism 7.0 was used for data analysis. Data are presented as mean ± SEM. Unpaired t-tests and multiple t-tests or one-way ANOVA followed by Dunnet's post hoc correction were used for comparisons between two or multiple groups, respectively. Significance was indicated as follows: *, p < 0.05; **, p < 0.01, and ***, p < 0.001.
Because super-enhancer can regulate the transcription of multiple proximate genes within a chromatin loop [6], we also tested the mRNA level of ITGB1BP1 located next to ASAP2 ( Figure 1F,G) and found a significant correlation between the genes before and after 1,25(OH) 2 D 3 stimulation ( Figure 1G). VSEs are dense clusters of enhancers identified by VDR binding regions. Our previous analysis suggested that [6] ASAP2 VSE (GRCh37/hg19 chr2: 9400523-9401668) was a 1,25(OH) 2 D 3 stimulation-dependent enhancer and could not be identified as a superenhancer without 1,25(OH) 2 D 3 stimulation, as evidenced by the ChIP-seq signal density of VDR binding in GSE89431 [25]. These VSEs in 1,25(OH) 2 D 3 -stimulated THP-1 cells were significantly enriched for the motifs of TFs VDR, RUNX3, and CEBPA [6]. Therefore, first, we validated VDR binding in the ASAP2 VSE region by ChIP assay (Figure 2A). Subsequently, we searched the binding sites of the TFs (i.e., VDR, RUNX3, and CEBPA) in the ASAP2 VSE region based on their motifs in the JASPAR database. Because RUNX1/2 and RUNX3 share similar motif sequences [26,27], we also searched for regions with RUNX1 and RUNX2 motifs. Interestingly, the top (i.e., the highest relative scores estimated by JASPAR) motif regions of VDR and RUNX3 were close to each other in the ASAP2 VSE, and the top motif regions of SPI1 (the key and pioneer TF of THP-1) and CEBPA (the pioneer TF of THP-1) were close to each other in the region ( Figure 2B). ChIP-seq data in GSE89431 [25] indicate that the VDR motif-enriched region between -640 and -420 and SPI1 motif-enriched region between -960 and -640 indeed show VDR ChIP-seq peak and SPI1 ChIP-seq peak, respectively ( Figure 2B).
To validate the importance of VDR and RUNX3 binding sites in the 640/420 core enhancer region, these sites were separately deleted by site-directed mutagenesis ( Figure 2E). All these deletions significantly reduced the corresponding luciferase activities of pGL4.23-640 ( Figure 2F).
Notably, the 960/640 inhibitory region had SPI1/CEBPA motifs. We speculated that the inhibitory 960/640 region locked the activity of 1,25(OH) 2 D 3 -responsive VSE/960 region, which could be liberated by stimulation with 1,25(OH) 2 D 3 , resulting in the whole activation of the VSE. To examine the inhibitory effect of CEBPA binding, we deleted the two CEBPA binding sites separately ( Figure 2G) and found that the first deletion of the CEBPA binding site enhanced the activity of VSE after 1,25(OH) 2 D 3 treatment ( Figure 2H).

The Cytoskeletal-Associated Proteins and Interactors of ASAP2
Furthermore, mass spectrometry was performed to identify the interactors of ASAP2 ( Figure 5A,B, Supplementary File Table S2) and enriched functions ( Figure 5C,D) in 1,25(OH) 2 D 3 -treated macrophages.
After GO and KEGG enrichment analyses for 191 potential interactors, seven proteins, including SPECC1L/FKBP15 with the top score (Protein Q-score > 8) and the cytoskeletal-associated RAC1, were found to be enriched in the annotation of "actin filament" (Figure 5C), consistent with the above results.
Notably, the cytoskeletal-associated functions (i.e., "intermediate filament cytoskeleton", "intermediate filament", and "actin filament") were only enriched for the interactors in THP M0 macrophages. Furthermore, 41 common ASAP2 interactors between THP-1 and THP-1 M0 ( Figure 6A) were identified and found to be enriched in the functions of RNA splicing, telomere maintenance/organization, and DNA metabolic process ( Figure 6D). Interestingly, these 41 interacting proteins, including SAMHD1, were enriched in type I interferon-mediated signaling pathway, a result reported herein for the first time.

ASAP2 Promoted Interferon Signaling and Anti-Virus-Associated Pathways
To distinguish the functions of ASAP2 between 1,25(OH)2D3-stimulated THP-1 monocytes and 1,25(OH)2D3-stimulated THP-1 M0 macrophage, we performed mass To examine the functions of ASAP2, RNA-seq analysis was performed for shASAP2 and shNC samples (Supplementary File Table S4, GSE217201). Knockdown of ASAP2 reduced the expression of genes involved in response to interferon (IFN) signaling and antivirus-associated pathways ( Figure 6E,F). By overlapping enriched genes in IFN signaling/ anti-virus functions obtained from GO and GSEA enrichment analyses, 45 IFN signaling/ anti-virus-associated genes (including SAMHD1) were identified and found to reduce following ASAP2 knockdown ( Figure 6G,H), suggesting the necessity of ASAP2 for IFN signaling/anti-virus-associated pathways. Interestingly, SAMHD1 was also enriched in the type I IFN-mediated signaling pathway as an ASAP2 interactor ( Figure 6D). Therefore, we performed an immunoprecipitation assay for ASAP2 and SAMHD1 in 1,25(OH) 2 D 3 -treated THP-1-derived macrophages and validated their interaction ( Figure 6I,J).

Vitamin D Reduced the Number of Apoptotic Cells in EAE and Promoted Macrophage Efferocytosis in Peritonitis without Changing the Total mRNA Level of Asap2
To examine the effect of vitamin D on efferocytosis and alteration of Asap2 expression in inflammatory diseases, we constructed a model for central nervous system (CNS) inflammation EAE and thioglycolate peritonitis.
Notably, the cytoskeletal-associated functions (i.e., "intermediate filament cytoskeleton", "intermediate filament", and "actin filament") were only enriched for the interactors in THP M0 macrophages. Furthermore, 41 common ASAP2 interactors between THP-1 and THP-1 M0 ( Figure 6A) were identified and found to be enriched in the functions of RNA splicing, telomere maintenance/organization, and DNA metabolic process ( Figure  6D). Interestingly, these 41 interacting proteins, including SAMHD1, were enriched in type I interferon-mediated signaling pathway, a result reported herein for the first time.
To examine the functions of ASAP2, RNA-seq analysis was performed for shASAP2 and shNC samples (Supplementary File Table S4, GSE217201). Knockdown of ASAP2 reduced the expression of genes involved in response to interferon (IFN) signaling and antivirus-associated pathways ( Figure 6E,F). By overlapping enriched genes in IFN signaling/anti-virus functions obtained from GO and GSEA enrichment analyses, 45 IFN signaling/anti-virus-associated genes (including SAMHD1) were identified and found to reduce following ASAP2 knockdown ( Figure 6G,H), suggesting the necessity of ASAP2 for IFN signaling/anti-virus-associated pathways. Interestingly, SAMHD1 was also enriched in the type I IFN-mediated signaling pathway as an ASAP2 interactor ( Figure 6D). Therefore, we performed an immunoprecipitation assay for ASAP2 and SAMHD1 in 1,25(OH)2D3treated THP-1-derived macrophages and validated their interaction ( Figure 6I,J). To examine the effect of vitamin D on efferocytosis and alteration of Asap2 expression Although we observed the modestly (not significantly) protective effect of vitamin D on EAE given the relatively lower EAE score ( Figure 7A) and higher body weight ( Figure 7B) in the vitamin D normal group compared to the vitamin-D-deficient group, inflammatory cell infiltration ( Figure 7C-E) and demyelination ( Figure 7F) showed no difference between the two groups. Notably, an increased number of apoptotic cells in the spinal cord of the vitamin D deficient group was observed by TUNEL staining (Figure 7G). We also tested the mRNA level of Asap2 in brain mononuclear cells, which showed no difference between vitamin D normal and -deficient groups ( Figure 7H). After analyzing the microarray data of human brain samples (GSE131282), the mRNA level of ASAP2 was found to increase in the lesions of gray matter from multiple sclerosis (MS) patients compared to the normal regions from MS patients or healthy individuals ( Figure 7I). Based on Protein Atlas and UCSC Cell Browser on single-nucleus RNA-seq data of MS lesions [39], the transcription of ASAP2 was mainly observed in the astrocytes ( Figure 7J-K), suggesting the potential cell specificity of ASAP2 in CNS for future reference.
R PEER REVIEW 17 of 23 7B) in the vitamin D normal group compared to the vitamin-D-deficient group, inflammatory cell infiltration ( Figure 7C-E) and demyelination ( Figure 7F) showed no difference between the two groups. Notably, an increased number of apoptotic cells in the spinal cord of the vitamin D deficient group was observed by TUNEL staining ( Figure 7G). We also tested the mRNA level of Asap2 in brain mononuclear cells, which showed no difference between vitamin D normal and -deficient groups ( Figure 7H). After analyzing the microarray data of human brain samples (GSE131282), the mRNA level of ASAP2 was found to increase in the lesions of gray matter from multiple sclerosis (MS) patients compared to the normal regions from MS patients or healthy individuals ( Figure 7I). Based on Protein Atlas and UCSC Cell Browser on single-nucleus RNA-seq data of MS lesions [39], the transcription of ASAP2 was mainly observed in the astrocytes ( Figure 7J-K), suggesting the potential cell specificity of ASAP2 in CNS for future reference.  We assessed the protective effect of 1,25(OH) 2 D 3 on peritonitis. Consistent with our hypothesis, although 1,25(OH) 2 D 3 obviously reduced the number of recruited immune cells in the peritoneum, the peritoneal macrophages showed higher efferocytosis efficiency for the injected PKH26-stained apoptotic thymocytes in 1,25(OH) 2 D 3 group ( Figure 8A,B). To elucidate the role of Asap2 underlying 1,25(OH) 2 D 3 -strengthened efferocytosis, we tested the mRNA level of Asap2 in all peritoneal immune cells before and after intraperitoneal (i.p.) injection of apoptotic thymocytes. The expressional level of Asap2 reduced modestly (not significantly increased) following 1,25(OH) 2 D 3 injection ( Figure 8C,D), mostly due to its complex spatio-temporal expression in vivo.

Discussion
This study attempted to elucidate the regulatory mechanism of VDR-bound enhancer on the transcription of ASAP2 in THP-1 cells and the potential effects of upregulated ASAP2 levels in THP-1-derived macrophages. We found three regulatory regions in the ASAP2 VDR-bound enhancer, which separately played the role of core enhancer, inhibition, and promotion for the transcription of ASAP2 after 1,25(OH)2D3 stimulation. 1,25(OH)2D3 could promote efferocytosis of apoptotic Jurkat cells by THP-1 M2 macrophages partly through the upregulation of ASAP2 expression and activation of RAC1. The previously unknown functions of ASAP2, including RNA splicing, RNA transport, mitochondrial complex, and anti-virus, were revealed for the first time by mass spectrometry and RNA-seq analyses. Both EAE and peritonitis models validated the promotive effect of vitamin D on efferocytosis in vivo, while the total mRNA level of ASAP2 remained unchanged. Our findings indicated the regulatory mechanism and potential functions of ASAP2, suggesting its potential as a treatment target against inflammatory diseases.
The direct and indirect regulation of vitamin D on its target genes has been extensively studied, suggesting a highly cell-specific manner of action, a significant overlap of biological process regulation in humans and mice, and the critical spatio-temporal vitamin D-driven super-enhancer regions in the context of chromatin [7][8][9]. VDR super-enhancer is a stretch of VDR binding regions with a high rank of VDR ChIP-seq signal intensity, potentially showing a strong enhancer activity for functional genes in specific cell

Discussion
This study attempted to elucidate the regulatory mechanism of VDR-bound enhancer on the transcription of ASAP2 in THP-1 cells and the potential effects of upregulated ASAP2 levels in THP-1-derived macrophages. We found three regulatory regions in the ASAP2 VDR-bound enhancer, which separately played the role of core enhancer, inhibition, and promotion for the transcription of ASAP2 after 1,25(OH) 2 D 3 stimulation. 1,25(OH) 2 D 3 could promote efferocytosis of apoptotic Jurkat cells by THP-1 M2 macrophages partly through the upregulation of ASAP2 expression and activation of RAC1. The previously unknown functions of ASAP2, including RNA splicing, RNA transport, mitochondrial complex, and anti-virus, were revealed for the first time by mass spectrometry and RNAseq analyses. Both EAE and peritonitis models validated the promotive effect of vitamin D on efferocytosis in vivo, while the total mRNA level of ASAP2 remained unchanged. Our findings indicated the regulatory mechanism and potential functions of ASAP2, suggesting its potential as a treatment target against inflammatory diseases.
The direct and indirect regulation of vitamin D on its target genes has been extensively studied, suggesting a highly cell-specific manner of action, a significant overlap of biological process regulation in humans and mice, and the critical spatio-temporal vitamin D-driven super-enhancer regions in the context of chromatin [7][8][9]. VDR super-enhancer is a stretch of VDR binding regions with a high rank of VDR ChIP-seq signal intensity, potentially showing a strong enhancer activity for functional genes in specific cell types. In addition to VDR, other transcription factors (i.e., RUNX3 and CEBPA) are enriched in the VDR super-enhancer regions, thus cooperating with VDR and regulating gene transcription. The RUNX family interacts with VDR in different cell types [26,27]. Herein, we showed that RUNX3 cooperated with VDR to upregulate the transcription of ASAP2 at the core enhancer and promoting regions in THP-1 cells. SPI1 (the key/pioneer TF of THP-1 [40,41]) and CEBPA (the pioneer TF for THP-1 [24,42]) cooperate to inhibit the transcription of ASAP2 at the inhibitory region of the VDR bound-enhancer. The mechanism of their inhibition on 1,25(OH) 2 D 3 -upregulated ASAP2 may be due to the changed spatial structure of the transcription loop and warrants further investigation. ARFGAP ASAP2 is associated with FcγR-mediated phagocytosis and vesicular transport. For the first time, we showed that the interactome of ASAP2 in THP-1 and the common interacting proteins between THP-1 and THP-1 M0 that were enriched in the RNA and DNA regulatory functions, e.g., RNA splicing, telomere maintenance, DNA metabolic process, and the type I interferon-mediated signaling pathway. Only in THP-1 M0, the ASAP2 interacting proteins were enriched in actin-associated functions. Among these functions, ASAP2 not only interacted with proteins in the IFN signaling pathway but also regulated the expression of genes in the IFN signaling pathway and antiviral immunity. SAMHD1, a dNTP triphosphohydrolase (dNTPase), can hydrolyze intracellular dNTPs, thereby reducing viral reverse transcription and cDNA synthesis [43,44]. Vitamin D can also promote antibacterial and antiviral innate immunity by inducing antimicrobial peptides, lowering intracellular iron concentration, and enhancing autophagy [45,46]. Previously, the association between IFN signaling/anti-virus immunity and other ARFGAPs has been identified. For example, ARFGAP Domain-Containing Protein 2 (ADAP2) can mediate IFN responses, promote type I IFN production, and restrict the entry of RNA viruses [47,48].
Because ASAP2 is a risk gene associated with MS risk SNP rs1109670 located upstream [49], we first tested the effect of vitamin D on the EAE model. However, herein, we did not find a significant protective effect of normal vitamin D on CNS inflammation, which was potentially due to the attenuated antigen-presenting ability within peripheral lymph nodes in the vitamin D deficient group. Although the positive correlation between vitamin D status and MS and the protective effect of vitamin D on EAE mice has been confirmed [4,50], some studies show lower or unchanged EAE clinical scores in the vitamin-D-deficient group compared to the normal group, potentially due to the weakened antigen-presenting ability in peripheral lymph nodes [51][52][53][54]. Herein, we showed the modestly protective effect of vitamin D on EAE and its potential promoting effect on efferocytosis due to the fewer apoptotic cells in the spinal cord of the normal vitamin D group, suggesting the potentially important role of efferocytosis in MS. However, we did not observe upregulated ASAP2 levels in the vitamin D normal group and needs further validation in specific cell types (macrophage/microglia/astrocytes) or pathological sections (normal appearing/lesion regions) of the CNS in the future.
The inhibitory effect of vitamin D on peritoneal dialysis-related peritonitis, bacterial peritonitis, and zymosan-induced peritonitis has been reported previously [55][56][57] given its regulatory ability on innate immune cells by increasing anti-infectious effect and resolving inflammation. Moreover, upregulated IL-10 levels in regulatory T (Treg) cells can enhance efferocytosis and further attenuate peritoneal inflammation [36]. Herein, we showed that active vitamin D 3 could increase the efferocytosis of macrophages in thioglycolate-induced peritonitis. However, we did not observe any alterations in ASAP2, suggesting stable expression of ASAP2 or potential time-dependent upregulation during peritonitis.
In summary, we identified different regulatory regions of VDR-bound enhancer in ASAP2 and the promotive effect of 1,25(OH) 2 D 3 /ASAP2/RAC1 axis on efferocytosis, thus suggesting its potential as a new therapeutic target against inflammatory diseases. Several new functions of ASAP2 in macrophages were identified by mass spectrometry and RNAseq analyses. The interaction between ASAP2 and SAMHD1 in the regulation of the IFN signaling pathway and antiviral immunity needs further studies for validation as a potential antiviral target.