RNA-seq of THP-1 and U937 exposed to enamel matrix derivative

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Miron, Reinhard Gruber This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7240030/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Objectives: Enamel matrix derivatives (EMDs) are applied in periodontal defects and gingival recession, a process where macrophages contribute to the clinical outcome. There is a need for standardized bioassays to better understand and monitor how EMD affects macrophages in vitro. Materials and methods: We propose using THP-1 and U937, both widely established monocytic cell lines, as bioassays in EMD research. Both cell lines have different origins, as THP-1 is a leukemia cell line, and U937 originates from the pleural effusion of a patient with histiocytic lymphoma. To understand their differential response to EMD, we employed an RNA-seq approach revealing changes in the genetic signatures of THP-1 and U937 cells. Results: When applying a threshold of 1.5 log2 fold-change and a significance of 2.0-log10, we could identify 5/37 and 30/23 up- and down-regulated genes in THP1 and U937 cells, respectively. In THP-1, the upregulated genes included S100A8, S100A9 and CD38; downregulated gene included ADM, CD48, IL24, MMP1, and PDGFB. In U937, most striking was the increase of alpha subunit integrins ITGA1, ITGA2, ITGA6, and the decrease of genes including OLR1, CCL1, CCL4L2, CCL8, IL21R, MMP7, PDGFB and MMP25. We further show that the TGF-β receptor type I kinase inhibitor SB431542 blocked the expression changes of S100A8, S100A9, CD38, ITGA2, ITGA6, and OLR1 but failed to reverse PDGFB. Conclusions: These data serve as a primer for developing macrophage bioassays to measure EMD activity in the context of TGF-β signaling. Clinical relevance : To identify a panel of genes, ideally being strongly regulated by EMD, in established THP1 and U937 cell lines, with a potential clinically relevant function in periodontal and peri-implant regeneration. Clinical trial number: Not applicable. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Periodontal research is driven by the global health burden associated with periodontitis. This local chronic inflammation causes catabolic changes in the tooth-supporting tissues and progresses if left untreated, ultimately leading to tooth loss [ 1 ]. The same applies to peri-implantitis, where chronic inflammation causes inflammatory osteolysis and implant loss [ 2 , 3 ]. Apart from preventing the onset of local inflammation through adequate oral hygiene, when treatment becomes necessary, first clean the periodontal pockets and then apply methods and devices supporting the attachment, sealing off the space towards the oral cavity [ 4 , 5 ]. Similar attempts are made to regain peri-implant health [ 3 ]. When devices are applied, it is essential to understand the respective local cellular response, particularly when the treatment is clinically successful. One commercially available device is Emdogain® (enamel matrix derivative, EMD), produced by Biora, a Swedish company, and distributed by Straumann Biologics Division [ 5 , 6 ]. EMD is derived from the developing tooth germs of 6-month-old piglets and applied in a propylene glycol alginate vehicle solution [ 6 ]. Clinically, meta-analysis summarized the use of EMD, for instance, in the treatment of intrabony defects [ 5 , 7 , 8 ] or flap surgery [ 5 , 9 ]. Support also came from preclinical research on attachment formation after tooth transplantation [ 10 ] and bone healing around titanium implants [ 11 ], as well as the formation of acellular cementum [ 12 ]. The clinical effects of EMD were attributed to its main component, the amelogenins [ 6 , 13 ], which is why human recombinant amelogenin was proposed for periodontal healing in intrabony defects [ 14 ]. Accumulating evidence further suggests that EMD exhibits TGF-β activity, as the cellular responses could be at least partially attributed to this signaling pathway. Insights into the cellular mechanisms were gained from mesenchymal lineage cells, such as oral fibroblasts. EMD exerts a robust change in the transcriptome, which was significantly diminished by SB431542, a TGF-β receptor type I kinase inhibitor [ 15 – 17 ]. When considering hematopoietic lineage cells, the TGF-β activity of EMD mediates at least part of its anti-inflammatory activity in murine RAW 264.7 macrophages [ 18 , 19 ]. Additionally, in human blood cells and murine bone marrow cells, EMD was utilized to investigate osteoclastogenesis [ 20 – 23 ], with a particular focus on TGF-β signaling [ 22 ]. There is also support for amelogenin to suppress MHC II expression [ 24 ] and to promote M2 macrophage polarization [ 25 ] in U937 macrophages as well as in peripheral blood-derived macrophages. These studies targeted aspects of how EMD affects hematopoietic lineage cells; however, they were not based on a whole genome transcriptomic approach in established bioassays. THP-1 and U937 are macrophage cell lines commonly used for bioassays. THP-1 was isolated from the peripheral blood of an acute monocytic leukemia patient [ 26 ], while U937 cell was derived from the pleural effusion of a patient with histiocytic lymphoma [ 27 ]. Furthermore, THP1 [ 28 , 29 ] and U937 [ 30 , 31 ] cells are widely utilized to investigate the inflammatory response to LPS. This pathogen-associated molecular pattern binds to toll-like receptors, especially when combined with interferon-gamma (IFNγ). Both cell lines respond to amelogenin [ 24 ] and TGF-β. For instance, TGF-β weakly increased CD163, VEGF, and LFA-1 and decreased CD86 [ 32 , 33 ]. TGF-β1 also increased the resistance of U937 cells to apoptotic cell death [ 34 ]. Thus, there is reason to assume that THP-1 and U937 cells might respond to EMD and its TGF-β activity in vitro. However, no panel of highly regulated genes is currently available to establish a bioassay that further serves the purpose of studying the involvement of TGF-β signaling. The overall aim of the present study was, therefore, to identify genes strongly regulated in EMD-exposed THP-1 and U937 cells based on bulk RNA-seq analysis and, based on these findings, establish an initial gene panel that allows testing for the possible involvement of TGF-β signaling to mediate the activity of EMD by blocking TGF-β receptor type I kinase with the inhibitor SB431542. Materials and Methods THP-1 and U937 cell lines The THP-1 leukemia cell line and U937 cell line derived from the pleural effusion of a patient with histiocytic lymphoma (ATCC, TIB-202 and CRL-1593.2) were expanded in RPMI 1640 medium (Gibco Life Technologies, CA, USA) containing 10% FCS (Bio&Sell GmbH, Nuremberg, Germany) and 1% antibiotics in a humidified incubator at 37°C with 5% CO 2 . To drive the cells towards a macrophage’s lineage, the cell lines exposed to 10 ng/mL phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich, St. Louis, MO) for 48 hours. Then, the THP-1 and U937 macrophage-like cells were exposed to 300 µg/mL Emdogain® (EMD; Straumann Group, Basel, Switzerland) or serum-free medium for 24 hours before RNA extraction. In indicated experiments, 10 µM of the TGF-β receptor I kinase inhibitor SB431542 (Calbiochem, Merck, Billerica, MA, USA) was used. In addition, indicated experiments were performed using positively selected CD14 + cells from whole blood (StraightFrom Whole Blood, in combination with the autoMACS Separators, Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany), thereby avoiding differentiation into macrophages. Total RNA isolation, sequencing, and data analysis Total RNA (ExtractMe total RNA kit, Blirt S.A., Gdańsk, Poland) was sued to prepare sequencing libraries at the Core Facility Genomics of the Medical University of Vienna, using the QuantSeq 3' FWD protocol version 2 with unique dual indices (Lexogen GmbH, Vienna, Austria). A QC-checked was done with a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) and quantified using Qubit dsDNA HS Assay (Invitrogen, Waltham, MA). The pooled libraries were sequenced on a P2 flowcell on a NextSeq2000 instrument (Illumina, San Diego, CA) in 1x75bp single-end sequencing mode. FASTQ fines were generated using the Illumina bcl2fastq command-line tool (v2.19.1.403) and the Lexogen idemux tool for optimal demultiplexing of long, unique, dual indices. Reads were cropped and filtered using cutadapt version 2.8 to trim polyA tails, remove reads with N's, and trim bases with a quality of less than 30 from the 3' ends of the reads [ 35 ]. Fastq files were aligned to the human reference genome version GRCh38 with Gencode 29 annotations using the STAR aligner [ 36 ] version 2.6.1a in 2-pass mode. STAR counted raw reads per gene. Differential gene expression was computed using DESeq2 version 1.22.2 [ 37 ]. The datasets generated during the current study are available in the Gene Expression Omnibus (GEO) repository, under the accession numbers GSE307844. Differentially genes with an adjusted p-value of 0.05 are shown in Supplement Files 1. Reverse Transcription Quantitative Real-Time PCR (RT-qPCR) Total RNA was transcribed into complementary DNA (LabQ, Labconsulting, Vienna, Austria) followed by a amplification in the presence of master mix (LabQ, Labconsulting, Vienna, Austria) and primers (Supplement Files 2) on a CFX Connect™ Real-Time PCR device (Bio-Rad Laboratories, Hercules, CA). Expression changes were normalized to GAPDH by the ΔΔCt method and further towards unstimulated controls. Volcano plot, Venn diagram, heat map, protein-protein interactions, and gene set enrichment analysis. The web-based VolcaNoseR was used to prepare the volcano blots with minimum log2 2.5-fold change and a minus log10 significance level of 2.0 [ 38 ]. Also, we based, Venn diagrams were prepared with InteractiVenn [ 39 ], and Morpheus ( https://software.broadinstitute.org/morpheus ) to show heat maps. The STRING database systematically collects and integrates protein-protein interactions- both physical interactions and functional associations [ 40 ]. For gene enrichment analysis, we took advantage of the g: Profiler online tool [ 41 ]. Results Principal component analysis (PCA) of gene expression changes by EDM-activated THP-1 and U937 Here, we examined the impact of EMD on the global gene expression profiles of THP-1 and U937 cells using bulk RNA sequencing. Principal component analysis revealed that the first principal component explained most of the variance between the THP-1 and U937 cell lines, and the second principal component visualized the changes caused by the EMD treatment, which were more homogeneous but less pronounced in U937 cells compared to THP-1 cells (Fig. 1 , Supplement Fig. 3 ). The plot shows that THP1 and U937 data are distinguished in the PC 1 while the effects of EMD are visible in PC2. There is a heterogenicity caused by one data point on THP1 controls. Volcano analysis of gene expression changes by EMD-exposed THP-1 and U937 cells. To understand the direction and magnitude of gene expression changes a Volcano plot was prepared (Fig. 2 ). The volcano plot represents a minimum 2.0-log10 significance and a 1.5-log2 fold- expression change. Based on these criteria, we recognized 5 and 30 upregulated genes in THP-1 and U937 cells, respectively. Also, 37 and 23 down-regulated genes were identified (Fig. 3 ). In the THP-1 cells, the most obvious was the increased expression of S100A8, S100A9, and CD38, as well as the decrease in GINS4, GEM, RRM2, CAV1, ADM, CD48, and PDGFB. In the U937 cells, ITAG1, ITGA2, ITAG6, OLR1, and FN1 were apparently increased, while PDGFB, besides CCL1 and CCL8, were among the decreased genes. The volcano plot further highlights the differential response of the THP1 and U937 cells to EMD treatment and details are depicted in Supplement Files 4. Heat map of gene expression changes by EMD-exposed THP-1 and U937 cells Next, we showed the heat map of the 5/37 (42) and 30/23 (53) genes selected by the indicated thresholds in THP-1 and U937 cells. Figure 3 highlights the variance and magnitude of the differential gene expression. The heat map further confirms the heterogeneity within the cell response of THP-1 compared to U937 cells when exposed to EMD. Moreover, consistent with the PCA, one sample of the THP1 down-regulated genes resembles an outlier, and the data should be interpreted accordingly. The underlying data are summarized in Supplement Files 5. Venn analysis of genes regulated by EMD-exposed THP1 and U937 cells Next, a Venn analysis was performed on the differential response of THP-1 and U937 cells to EMD stimulation (Fig. 4 , Supplement File 6). Venn analysis identified only two commonly regulated genes, with PDGFB being decreased in both THP-1 and U937 cells, whereas RIPOR2 was increased and decreased in the respective cell lines. This analysis further demonstrates the heterogeneity of how THP-1 and U937 cells respond to EMD. This analysis is restricted to the genes selected under a 1.5 log2 fold-change and a significance of 2.0 log10. Among the 42 and 53 significantly regulated genes in THP-1 and U937 cells, they have only two genes in common that EMD regulates. STRING analysis of expression changes in THP-1 and U937 cells exposed to EMD. The STRING analysis was performed to identify potential clusters of gene expression changes and details are summarized in Supplement File 7. THP1 cells showed eight clusters, highlighting “Regulation of neutrophil degranulation” (CD38, CD48, SLC12A8, SYK, ITGAM, C3AR1) and “Neutrophil aggregation” (S100A8, S100A9). In the U937 cells, a significant cluster was related to "ECM-receptor interaction” (ITGA1, ITGA2, ITGA6, MMP3, MMP7, MMP25, IL7R, IL21R, FAP, SPP1, FN1, PROCR, and PDGFB) and “Monocyte chemotaxis” (CCL1, CCL20, CCL4L2). (Fig. 5 ). The protein–protein association network and functional enrichment analyses of THP-1 (A) and U937 (B) cells, based on differentially expressed genes 42 and 53, are depicted. G: Profiler analysis of gene expression changes in THP-1 and U937 cells by EMD Following was a functional enrichment analysis of the 42 genes that were strongly regulated (5 up-regulated and 37 down-regulated genes) in THP-1 cells (Figure 6). Gene ontology confirmed the expected enrichment for Toll-like receptor binding (GO:0035325) linking S100A8, S100A9, and SYK. Enrichment was also achieved for leukocyte migration (GO:0050900) with S100A8, S100A9, PDGFB, SYK, SPNS2, APOD, CYP19A1, EDN1, HSD3B7, C3AR1, JAM2 and RIPOR2. As shown in Figure 7 and in Supplement Files 8, the 53 regulated genes in U937 cells exhibited enrichment for chemokine activity (GO:0008009), specifically with CCL1, CCL8, CCL4L2, and CCL20. Also highlighted is collagen binding (GO:0005518) with ITGA1, ITGA2, FN1 and PDGFB. Moreover, we have collagen catabolic process (GO:0030574) including MMP3, MMP7, MMP25, FAP and integrin complex (GO:0008305) with ITGA1, ITGA2 and ITGA6. Functional enrichment analysis (over-representation analysis (ORA) or gene set enrichment analysis) was performed using the g: Profiler online tool. The top significant pathways were highlighted and labeled numerically. The p-value was adjusted (Padj) for multiple testing using the Benjamini-Hochberg method. Functional enrichment analysis (over-representation analysis (ORA) or gene set enrichment analysis) was performed using the g:Profiler online tool. The top significant pathways were highlighted and labeled numerically. Using the Benjamini-Hochberg method, the P value was adjusted (Padj) for multiple tests. RT-PCR analysis of selected genes in THP-1 and U937 cells exposed to EMD and SB431542 A RT-PCR analysis of selected genes was used to confirm the RNAseq analysis and to support the notion that cell responses to EMD are linked to TGF-β activity. EMD exposure of THP-1 and U937 cells caused the expected increase in S100A8, S100A9, CD38, and ITGA2, ITGA6, and OLR1 expression, respectively; however, not in the presence of the TGF-β receptor type I kinase-inhibitor SB431542 (Figure 8 and Supplement Files 8). The exception was that the decrease of PDGFB expression could not be reversed by SB431542, suggesting an expression change independent of TGF-β signaling (Figure 9). When freshly prepared primary CD14+ monocytes were exposed to EMD, CCL20 increased at least 3-fold; however, no significant changes were noticed for S100A8, S100A9, CD38, ITGA2, ITGA6, and OLR1 (data not shown). Discussion EMD is a tool used to facilitate periodontal and peri-implant regeneration, inspiring preclinical research on translating clinical endpoints into cellular responses [ 5 , 6 ]. EMD is initially derived from the tooth germs of piglets and thus represents a broad spectrum of proteins and possibly other bioactive components; a complex cellular response can be expected. Accumulating evidence suggests that, apart from mesenchymal cells [ 15 – 17 ], hematopoietic cells are also targeted by EMD, but this has not been tested on THP-1 and U937 cells. Apart from the functional in vitro bioassays related to the differentiation of hematopoietic cells towards macrophages [ 18 , 19 ] and osteoclasts [ 20 – 23 ], we lack bioassays to evaluate the expression of highly regulated genes in response to EMD. These genes reflect the functional response of macrophages to EMD and may further support the estimation of the biological activity of the various batches of EMD in the production process. Thus, our overall motivation was to employ an RNA-seq approach to identify a panel of genes that are ideally strongly regulated by EMD in established THP1 and U937 cell lines, with a potential clinically relevant function in periodontal and peri-implant regeneration. One main finding of the present research was that only five genes were upregulated in THP1 cells, considering the respective threshold levels. The upregulated genes in THP-1 cells were S100A8, S100A9, CD38 and PIK3IP1 [ 42 ], RIPOR2. These findings align with those of HSC-3 carcinoma cells exposed to EMD, which upregulate S100A8 and S100A9 [ 43 ], both members of the S100 calcium-binding protein family. These proteins are commonly expressed in macrophages, where they exert their proinflammatory and immunosuppressive properties [ 44 ]. S100A8 and S100A9 in saliva and gingival crevicular fluid have been proposed for screening established periodontitis [ 45 , 46 ]. Inflammatory mediators, such as TNF-α, increase their production in THP-1 cells [ 47 ]. Single-cell analysis revealed a macrophage subset expressing high levels of S100A8 and S100A9 in early gastric cancer tissues [ 48 ]. In addition, CD38, a multifunctional transmembrane glycoprotein, is also increased in THP-1 cells, for instance by IFN-γ[ 49 ], erythritol [ 49 ], and retinoic acid, a vitamin A metabolite [ 50 ]. Daratumumab and Isatuximab, both antibodies binding to CD38, were granted therapy drug status for multiple myeloma [ 51 ]. Considering the clinical relevance and the robust increase by EMD, the expression of S100A8, S100A9, and CD38 in THP1 cells is one of the proposed bioassay markers. Another key finding was that EMD caused 37 genes to be down-regulated in THP-1 cells. Among the downregulated genes is PDGFB, which has also been observed in U937 cells. Support for this finding comes from observations that IFN-γ treatment of THP-1 cells caused the decreased expression of PDGFB [ 52 ]. Moreover, macrophage-derived PDGFB induces smooth muscle cell expansion, which may exceed pathological conditions, such as in pulmonary hypertension [ 53 ] and obesity-associated metabolic stresses, as it plays a significant role in regulating pericyte behavior during angiogenesis [ 54 ]. However, the protective role of macrophage-derived PDGF-BB on nucleus pulposus cells was also shown [ 55 ]. Down-regulated genes further include MMP1, which is usually not expressed in THP1 cells but is induced by PMA stimulation [ 56 ]. THP-1 expression of MMP-1 was associated with aldosterone-induced macrophage infiltration [ 57 ]. Significantly higher MMP-1 expression levels were found in the inflamed gingiva compared with healthy biopsies, and Filifactor blockade caused an upregulation of MMP-1 in THP-1 cells [ 58 ], suggesting a potential bioassay where EMD can be tested for modulation of high MMP-1 levels. Additionally, the levels of MMP-1 were higher at sites with periodontitis and peri-implantitis compared to sites with healthy teeth and implants [ 59 ]. However, there are further down-regulated genes that need attention. EMD also diminishes AMD in THP-1 cells, which are known to be producers of ADM [ 60 , 61 ]. However, the secretion of TNF-α in LPS-stimulated THP-1 cells was reduced in the presence of recombinant AMD [ 62 ]. Thus, AMD is considered a multifaceted peptide implicated in vasodilation, hormone secretion, antimicrobial defense, cellular growth, angiogenesis, and, importantly, chronic pain [ 63 ]. It was also identified in human gingival crevicular fluid [ 64 ]. Most strongly decreased by EMD is GINS4, a promoter of the cell cycle, but little is known about its impact on ferroptosis [ 65 ]. Moreover, partial loss-of-function mutations in GINS4 lead to NK cell deficiency with neutropenia [ 66 ]. Considering THP-1 is a tumor cell, the lowering of GINS4 by EMD may also serve as a bioassay in oncology; however, its clinical implication should be interpreted carefully. In addition, CAV1 is increasingly expressed in THP-1 cells by agonists of peroxisome proliferator-activated receptor gamma activation [ 67 ], and CAV1 expression is lower in leukemia cancer cell lines, including THP-1, when compared with normal white blood cells [ 68 ]. CAV1 partially mediates the role of gingipanins to permeabilize the blood-brain barrier [ 69 ], and CAV1 knockout mice exhibit increased bone size and stiffness. Strong CAV1 immunoreactivity was detected in gingival epithelial and endothelial cells [ 70 ]. Although a series of downregulated genes was identified, our THP-1 data revealed a larger variation; therefore, further refinement is needed in the selection of downregulated genes driven by EMD. In the U937 cells, 30 upregulated genes by EMD reached the threshold. Most impressively, and by the cluster analysis, is the significant increase in the expression of integrins: ITGA1, ITGA2, and ITGA6 by EMD. These transmembrane α integrins dimerize with β integrins, together serving as regulators of cell–matrix and cell-cell interaction. For example, α1β1 and α2β1 were identified as collagen receptors [ 71 ]. ITGA6 is effective in laminin binding. Integrin binding is not passive as it promotes outside-in signaling, thus affecting cell behavior [ 72 ]. ITGA2 and FN1 are ERK-dependent targets of TGF-β signaling [ 73 ]. The present data thus suggest that EMD-exposed U937 cells preferentially adhere to collagen- and laminin-rich ECM. This observation may lead to the establishment of an in vitro bioassay for cell adhesion to coated surfaces or biomaterials. Clinically, an increased expression of integrins in macrophages has potential relevance, for instance, in facilitating migration into a defect site and enabling local adhesion to the ECM [ 74 ]. Likewise, lectin-like oxidized low-density lipoprotein receptor 1, encoded by OLR1, is enriched in a proinflammatory subset of macrophages [ 75 ], together with CD36 [ 76 ]. Functional assays may, therefore, test the impact of EMD on macrophage binding and internalization of oxLDL. Besides, IL7R should be mentioned in the context of macrophage infiltration under inflammatory conditions [ 77 , 78 ], and MMP3 degrades various collagen types and other components of the ECM and activates other MMPs [ 79 ]. CCL20, which is primarily chemotactic for lymphocytes, was increased in macrophages following aortic dissection [ 80 ] and upon the polarization of THP1 cells [ 81 ] and U937 cells [ 82 ]. Our preliminary experiments with EMD-exposed CD14 + blood-derived monocytes consistently showed a robust increase in CCL20 expression, further supporting our attempt to initiate future research on RNA-seq with primary monocytes. U937 cells responded by 23 downregulated genes by EMD. For instance, EMD decreased the expression of the proteases MMP7 and MMP25. MMP7 is effective in cleaving casein, various collagen types, fibronectin, and proteoglycans, and MMP25 belongs to the membrane-type MMP (MT-MMP) subfamily [ 83 ]. It is required for a proper innate immune response, such as to bacterial LPS, hypergammaglobulinemia, and reduced secretion of proinflammatory molecules [ 84 ]. Additionally, IL21R is required for infiltrating macrophages following infection [ 85 ], and it also reduces the LPS-mediated secretion of inflammatory cytokines in macrophages [ 74 ]. Concerning the decrease of the chemokines, CCL1 is involved in atherosclerosis [ 86 ] and fibrosis [ 87 ]. CCL8 was upregulated in the periodontal ligament during the initial stage of OTM [ 88 ] and has been shown to impair muscle regeneration [ 89 ]. Macrophages with high CCL4L2 expression were identified in response to pathogenic bacteria in the intestine [ 90 ] and rheumatoid arthritis [ 91 ]. For review of chemokines in dentistry, see [ 92 ]. Moreover, potentially relevant for establishing a bioassay are SIGLEC1, an endocytic receptor that mediates clathrin-dependent endocytosis [ 93 ], and LILRB2, a class I MHC antigen receptor [ 94 ]. F2RL3, a receptor for activated thrombin or trypsin coupled to G proteins, is interesting [ 95 ], and CYSLTR1, a receptor for cysteinyl leukotrienes [ 96 ]. Apart from PDGFB, the selection of the gene set for a bioassay depends on the research question. We have recently exposed PBMCs to EMD in THP-1 cells [ 97 ]. Only FN1 and SERPINA1 are commonly upregulated genes, and PNOC is downregulated. In U937 cells, however, we have noticed that EMD also upregulates ITGA1, FN1, OLR1, SE-MA3C, SLC11A1, SNX10, TMEM52B, ALCAM, MS4A7, MS4A14, NMB, MRC2, and lowers MMP25, GDF15, SQLE, CD300A, and PTGIR – similar to the effects observed in U937 cells. However, PBMCs represent cells, including the monocyte phenotype, with CD14 + staining, while THP-1 and U937, due to their pre-differentiation, should be considered as macrophages. Interestingly, in PBMCs, EMD upregulates PDGFB [ 97 ]. However, PBMCs are a heterogeneous cell population, and we cannot differentiate between the cell types – whether the overlapping expression changes caused by EMD are caused by monocytes, granulocytes, or lymphocytes. We are currently exploring this aspect using a single-cell RNA-seq approach, and our preliminary data have at least identified OLR1 and PDGFB as being increasingly expressed in the monocyte fraction of whole blood exposed to EMD, respectively. Thus, our data suggest that the U937 cell line is preferable for reflecting the response of primary blood cells. There is also a debate about whether the activity of EMD is related to amelogenins, as they play a role in tooth development. Evidence for the role of amelogenin comes from knockout mice, which show a progressive deterioration of cementum linked to increased osteoclastogenesis [ 98 ]. Moreover, mice lacking ameloblastin experience severe enamel hypoplasia [ 99 ]. These genetically modified mouse models elegantly underscore the critical importance of amelogenin and ameloblastin during tooth development; however, insights into tooth development should not be extrapolated to their function after tooth eruption, and evidence for the clinical activity of EMD is, if at all, indirect. New insights may derive from generating inducible amelogenin and ameloblastin transgenic mouse models, which could reveal the role of amelogenins after tooth eruption. This model may simulate the local application of EMD. Today, we know that molars from M180 amelogenin transgenic mouse lines are similar to those of wild-type teeth [ 100 ]. Future in vitro research should compare the cell response to EMD with recombinant amelogenin and ameloblastin, as well as TGF-β – potentially using single cell RNAseq approach of whole blood or simply a bulk RNAseq of U937 or CD14 + monocytes, and, also relevant, oral fibroblasts – to identify to which extend the gene regulation caused by EMD is a consequence of amelogenesis and TGF-β activity, respectively. Finally, we have selected a panel of genes for THP-1 and U937, not only to conform to some of the RNA-seq data, but, perhaps more importantly, to gain first insights into the role of TGF-β signaling in the cell lines that mediates the activity of EMD. In support of previous studies in mesenchymal and murine hematopoietic cells, SB431542 significantly blocked the effect of EMD in raising the expression of S100A8 in THP-1 and ITGAs in U937 cells. Nevertheless, when focusing on the decreased expression of PDGFB, SB431542 failed to block the EMD effects significantly. Therefore, it is worthwhile to conduct future studies to determine the extent to which SB431542 blocks the spectrum of EMD-regulated genes and to explore whether other bioactive components in EMD may contribute to the changes in PDGFB and possibly other genes. Additionally, there is a demand for studies comparing primary human monocytes or macrophages with the THP-1 and U937 cell responses, with the overall aim of narrowing down the panel of genes that represent primary cells while taking advantage of a reproducible bioassay situation. Declarations Author Contributions : Conceptualization, L.P. and R.G.; data curation, L.P.; formal analysis, L.P. and R.G.; methodology, L.P.; project administration, L.P. and X.H.; supervision, L.P. and R.G.; validation, L.P.; visualization, L.P.; writing—original draft preparation, L.P. and R.G.; writing—review and editing, L.P., X.H., and R.G. All authors have read and agreed to the published version of the manuscript. Funding : The University Clinic of Dentistry has supported this study. L.P. and R.G. received funds from the ITI Projects 1729-2023 and 1709-2022. Data Availability : The datasets generated during the current study are available in the Gene Expression Omnibus (GEO) repository, under the accession numbers GSE307844. Differentially genes with an adjusted p-value of 0.05 are shown in Supplement File 1. All other data to generate the Figures are attached as Supplement Files 2-8. Acknowledgments : We thank the Core Facility Genomics of the Medical University of Vienna for performing the RNA-seq analysis, and especially Sophia Derdak for her valuable support. EMD was a kind gift from Straumann Österreich (Vienna, Austria). Conflicts of Interest : The authors declare no conflict of interest. Ethics approval and consent to participate: The study was conducted by the principles outlined in the Declaration of Helsinki. 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J Biol Chem. 2003;278:35743–8. 10.1074/jbc.M306284200 . Additional Declarations No competing interests reported. Supplementary Files SupplementFiles.zip Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 21 Oct, 2025 Reviewers invited by journal 09 Oct, 2025 Editor invited by journal 15 Sep, 2025 Editor assigned by journal 15 Sep, 2025 Submission checks completed at journal 12 Sep, 2025 First submitted to journal 12 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7240030","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":532904849,"identity":"b088c9c6-e9b2-4a98-ab3d-f93f69b91cfe","order_by":0,"name":"Layla Panahipour","email":"","orcid":"","institution":"University Clinic of Dentistry, Medical University of Vienna","correspondingAuthor":false,"prefix":"","firstName":"Layla","middleName":"","lastName":"Panahipour","suffix":""},{"id":532904850,"identity":"1e51dfb2-3081-433d-8e8c-e9f8b622340c","order_by":1,"name":"Xiaoyu Huang","email":"","orcid":"","institution":"University Clinic of Dentistry, Medical University of Vienna","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyu","middleName":"","lastName":"Huang","suffix":""},{"id":532904851,"identity":"45ed1e57-bc72-4e5d-916b-9c0f75694016","order_by":2,"name":"Richard J. 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19:31:45","extension":"html","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":247384,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7240030/v1/60f06873f77d7e5090a9d841.html"},{"id":94137917,"identity":"3c73fffb-fdbb-433a-a029-38fc8aacaad1","added_by":"auto","created_at":"2025-10-22 19:23:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":63966,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePCA for differentially expressed genes in EMD-activated THP-1 and U937 cells.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe plot shows that THP1 and U937 data are distinguished in the PC 1 while the effects of EMD are visible in PC2. There is a heterogenicity caused by one data point on THP1 controls.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7240030/v1/33eafcdac9b121e95d81f2d9.png"},{"id":94137918,"identity":"62ced0cf-a3dd-4cbc-b3da-07f9a97a3031","added_by":"auto","created_at":"2025-10-22 19:23:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":231009,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eVolcano plot analysis showing the impact of EMD on gene expression in THP-1 and U937 cells. \u003c/em\u003eThe expression changes caused by EMD in THP-1 (upper panel) and (lower panel) U937 cells are marked as red dots (upregulated) and blue dots (downregulated) genes. The threshold of 2.0 log10 significance level and a 1.5 log2 fold-change is marked by the scattered lines.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7240030/v1/54cf51d2ed9081614e38670f.png"},{"id":94137921,"identity":"df70e969-a16f-4ce6-a6c1-7e15b9103623","added_by":"auto","created_at":"2025-10-22 19:23:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":217085,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHeat map analysis for differentially expressed genes in THP1 and U937 cells treated with LPS and IFN-γ\u003c/em\u003e. Red indicates high expression levels, while blue indicates low levels, represented by darker and lighter shades of red and blue. Genes with a 1.5 log2 fold-change and a significance of 2.0-log10 were included in this analysis.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7240030/v1/06b7cb4988bc1fa6bc911e5b.png"},{"id":94137920,"identity":"dd87daf9-6405-4371-8593-ca95e8049556","added_by":"auto","created_at":"2025-10-22 19:23:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":34204,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eVenn analysis of the genes expressed in THP-1 and U937 cells exposed to EMD.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis analysis is restricted to the genes selected under a 1.5 log2 fold-change and a significance of 2.0 log10. Among the 42 and 53 significantly regulated genes in THP-1 and U937 cells, they have only two genes in common that EMD regulates.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7240030/v1/32d0fc389d5c26bb6f04f6bc.png"},{"id":94137925,"identity":"87e0d1f0-e9a8-49c4-87a9-0fb8a5dffae0","added_by":"auto","created_at":"2025-10-22 19:23:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":207073,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSTRING analysis of up- and down-regulated genes in THP-1 and U937 cells exposed to EMD.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe protein–protein association network and functional enrichment analyses of THP-1 (A) and U937 (B) cells, based on differentially expressed genes 42 and 53, are depicted.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7240030/v1/17fcface19c47e75d8610d0a.png"},{"id":94137933,"identity":"fef3f956-d2b0-44e4-a5ec-0efdbf751a32","added_by":"auto","created_at":"2025-10-22 19:23:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":229720,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003egProfiler analysis of differentially expressed genes in THP-1 with EMD\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eFunctional enrichment analysis (over-representation analysis (ORA) or gene set enrichment analysis) was performed using the g: Profiler online tool. The top significant pathways were highlighted and labeled numerically. The p-value was adjusted (Padj) for multiple testing using the Benjamini-Hochberg method.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7240030/v1/0a130aaa015b67bd4131625a.png"},{"id":94139175,"identity":"1d4b8c92-875e-459c-b729-24140b787df2","added_by":"auto","created_at":"2025-10-22 19:31:44","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":204795,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003egProfiler analysis of differentially expressed genes in U937 with EMD\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eFunctional enrichment analysis (over-representation analysis (ORA) or gene set enrichment analysis) was performed using the g:Profiler online tool. The top significant pathways were highlighted and labeled numerically. Using the Benjamini-Hochberg method, the P value was adjusted (Padj) for multiple tests.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7240030/v1/e61171b51f99ffc5e9f354fc.png"},{"id":94137940,"identity":"fbb2a616-b9aa-4127-a4c3-c05a817587f6","added_by":"auto","created_at":"2025-10-22 19:23:44","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":192067,"visible":true,"origin":"","legend":"\u003cp\u003eRT-PCR analysis of selected upregulated genes in THP-1 and U937 cells exposed to EMD and SB431542. RT-PCR analysis of THP-1 (upper panel) and U937 (lower panel) cells exposed to EMD with and without the TGF-β receptor I kinase inhibitor SB431542. Data points represented four independent experiments. Data were normalized to untreated control cells with x-fold changes compared to the untreated cells. The analysis was based on a Ratio paired t-test.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7240030/v1/fd2731933f8230e96ceb1c1b.png"},{"id":94139181,"identity":"cbcedc7d-a245-4ab8-9759-14c3f998f72d","added_by":"auto","created_at":"2025-10-22 19:31:44","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":57707,"visible":true,"origin":"","legend":"\u003cp\u003eRT-PCR analysis of PDGFB in THP-1 and U937 cells exposed to EMD and SB431542. RT-PCR analysis of THP-1 (left) and U937 (right) cells exposed to EMD with and without the TGF-β receptor I kinase inhibitor SB431542. Data points represented four independent experiments. Data were normalized to untreated control cells with x-fold changes compared to the untreated cells. The analysis was based on a Ratio paired t-test.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7240030/v1/f6f4e50691b6d5ef66aaef1d.png"},{"id":94141224,"identity":"76655667-9218-4d43-9332-aa8c9d5013df","added_by":"auto","created_at":"2025-10-22 19:55:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1861335,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7240030/v1/e302c874-087f-4ee8-bdd9-265efe9a10d2.pdf"},{"id":94140293,"identity":"2b482ba0-b38f-4809-aab6-7b1b1f0139c4","added_by":"auto","created_at":"2025-10-22 19:39:44","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":8832142,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementFiles.zip","url":"https://assets-eu.researchsquare.com/files/rs-7240030/v1/97cf6ca02411455df325bbd3.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"RNA-seq of THP-1 and U937 exposed to enamel matrix derivative","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePeriodontal research is driven by the global health burden associated with periodontitis. This local chronic inflammation causes catabolic changes in the tooth-supporting tissues and progresses if left untreated, ultimately leading to tooth loss [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The same applies to peri-implantitis, where chronic inflammation causes inflammatory osteolysis and implant loss [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Apart from preventing the onset of local inflammation through adequate oral hygiene, when treatment becomes necessary, first clean the periodontal pockets and then apply methods and devices supporting the attachment, sealing off the space towards the oral cavity [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Similar attempts are made to regain peri-implant health [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. When devices are applied, it is essential to understand the respective local cellular response, particularly when the treatment is clinically successful. One commercially available device is Emdogain\u0026reg; (enamel matrix derivative, EMD), produced by Biora, a Swedish company, and distributed by Straumann Biologics Division [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eEMD is derived from the developing tooth germs of 6-month-old piglets and applied in a propylene glycol alginate vehicle solution [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Clinically, meta-analysis summarized the use of EMD, for instance, in the treatment of intrabony defects [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] or flap surgery [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Support also came from preclinical research on attachment formation after tooth transplantation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and bone healing around titanium implants [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], as well as the formation of acellular cementum [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The clinical effects of EMD were attributed to its main component, the amelogenins [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], which is why human recombinant amelogenin was proposed for periodontal healing in intrabony defects [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Accumulating evidence further suggests that EMD exhibits TGF-β activity, as the cellular responses could be at least partially attributed to this signaling pathway.\u003c/p\u003e\u003cp\u003eInsights into the cellular mechanisms were gained from mesenchymal lineage cells, such as oral fibroblasts. EMD exerts a robust change in the transcriptome, which was significantly diminished by SB431542, a TGF-β receptor type I kinase inhibitor [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. When considering hematopoietic lineage cells, the TGF-β activity of EMD mediates at least part of its anti-inflammatory activity in murine RAW 264.7 macrophages [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Additionally, in human blood cells and murine bone marrow cells, EMD was utilized to investigate osteoclastogenesis [\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], with a particular focus on TGF-β signaling [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. There is also support for amelogenin to suppress MHC II expression [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and to promote M2 macrophage polarization [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] in U937 macrophages as well as in peripheral blood-derived macrophages. These studies targeted aspects of how EMD affects hematopoietic lineage cells; however, they were not based on a whole genome transcriptomic approach in established bioassays.\u003c/p\u003e\u003cp\u003eTHP-1 and U937 are macrophage cell lines commonly used for bioassays. THP-1 was isolated from the peripheral blood of an acute monocytic leukemia patient [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], while U937 cell was derived from the pleural effusion of a patient with histiocytic lymphoma [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Furthermore, THP1 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and U937 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] cells are widely utilized to investigate the inflammatory response to LPS. This pathogen-associated molecular pattern binds to toll-like receptors, especially when combined with interferon-gamma (IFNγ). Both cell lines respond to amelogenin [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and TGF-β. For instance, TGF-β weakly increased CD163, VEGF, and LFA-1 and decreased CD86 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. TGF-β1 also increased the resistance of U937 cells to apoptotic cell death [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Thus, there is reason to assume that THP-1 and U937 cells might respond to EMD and its TGF-β activity in vitro. However, no panel of highly regulated genes is currently available to establish a bioassay that further serves the purpose of studying the involvement of TGF-β signaling.\u003c/p\u003e\u003cp\u003eThe overall aim of the present study was, therefore, to identify genes strongly regulated in EMD-exposed THP-1 and U937 cells based on bulk RNA-seq analysis and, based on these findings, establish an initial gene panel that allows testing for the possible involvement of TGF-β signaling to mediate the activity of EMD by blocking TGF-β receptor type I kinase with the inhibitor SB431542.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eTHP-1 and U937 cell lines\u003c/h2\u003e\u003cp\u003eThe THP-1 leukemia cell line and U937 cell line derived from the pleural effusion of a patient with histiocytic lymphoma (ATCC, TIB-202 and CRL-1593.2) were expanded in RPMI 1640 medium (Gibco Life Technologies, CA, USA) containing 10% FCS (Bio\u0026amp;Sell GmbH, Nuremberg, Germany) and 1% antibiotics in a humidified incubator at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. To drive the cells towards a macrophage\u0026rsquo;s lineage, the cell lines exposed to 10 ng/mL phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich, St. Louis, MO) for 48 hours. Then, the THP-1 and U937 macrophage-like cells were exposed to 300 \u0026micro;g/mL Emdogain\u0026reg; (EMD; Straumann Group, Basel, Switzerland) or serum-free medium for 24 hours before RNA extraction. In indicated experiments, 10 \u0026micro;M of the TGF-β receptor I kinase inhibitor SB431542 (Calbiochem, Merck, Billerica, MA, USA) was used. In addition, indicated experiments were performed using positively selected CD14\u0026thinsp;+\u0026thinsp;cells from whole blood (StraightFrom Whole Blood, in combination with the autoMACS Separators, Miltenyi Biotec B.V. \u0026amp; Co. KG, Bergisch Gladbach, Germany), thereby avoiding differentiation into macrophages.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTotal RNA isolation, sequencing, and data analysis\u003c/h3\u003e\n\u003cp\u003eTotal RNA (ExtractMe total RNA kit, Blirt S.A., Gdańsk, Poland) was sued to prepare sequencing libraries at the Core Facility Genomics of the Medical University of Vienna, using the QuantSeq 3' FWD protocol version 2 with unique dual indices (Lexogen GmbH, Vienna, Austria). A QC-checked was done with a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) and quantified using Qubit dsDNA HS Assay (Invitrogen, Waltham, MA). The pooled libraries were sequenced on a P2 flowcell on a NextSeq2000 instrument (Illumina, San Diego, CA) in 1x75bp single-end sequencing mode. FASTQ fines were generated using the Illumina bcl2fastq command-line tool (v2.19.1.403) and the Lexogen idemux tool for optimal demultiplexing of long, unique, dual indices. Reads were cropped and filtered using cutadapt version 2.8 to trim polyA tails, remove reads with N's, and trim bases with a quality of less than 30 from the 3' ends of the reads [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Fastq files were aligned to the human reference genome version GRCh38 with Gencode 29 annotations using the STAR aligner [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] version 2.6.1a in 2-pass mode. STAR counted raw reads per gene. Differential gene expression was computed using DESeq2 version 1.22.2 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The datasets generated during the current study are available in the Gene Expression Omnibus (GEO) repository, under the accession numbers GSE307844. Differentially genes with an adjusted p-value of 0.05 are shown in Supplement Files 1.\u003c/p\u003e\n\u003ch3\u003eReverse Transcription Quantitative Real-Time PCR (RT-qPCR)\u003c/h3\u003e\n\u003cp\u003eTotal RNA was transcribed into complementary DNA (LabQ, Labconsulting, Vienna, Austria) followed by a amplification in the presence of master mix (LabQ, Labconsulting, Vienna, Austria) and primers (Supplement Files 2) on a CFX Connect\u0026trade; Real-Time PCR device (Bio-Rad Laboratories, Hercules, CA). Expression changes were normalized to GAPDH by the ΔΔCt method and further towards unstimulated controls.\u003c/p\u003e\u003cp\u003e\u003cem\u003eVolcano plot, Venn diagram, heat map, protein-protein interactions, and gene set enrichment analysis.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe web-based VolcaNoseR was used to prepare the volcano blots with minimum log2 2.5-fold change and a minus log10 significance level of 2.0 [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Also, we based, Venn diagrams were prepared with InteractiVenn [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], and Morpheus (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://software.broadinstitute.org/morpheus\u003c/span\u003e\u003cspan address=\"https://software.broadinstitute.org/morpheus\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to show heat maps. The STRING database systematically collects and integrates protein-protein interactions- both physical interactions and functional associations [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. For gene enrichment analysis, we took advantage of the g: Profiler online tool [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003ePrincipal component analysis (PCA) of gene expression changes by EDM-activated THP-1 and U937\u003c/h2\u003e\u003cp\u003eHere, we examined the impact of EMD on the global gene expression profiles of THP-1 and U937 cells using bulk RNA sequencing. Principal component analysis revealed that the first principal component explained most of the variance between the THP-1 and U937 cell lines, and the second principal component visualized the changes caused by the EMD treatment, which were more homogeneous but less pronounced in U937 cells compared to THP-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Supplement Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe plot shows that THP1 and U937 data are distinguished in the PC 1 while the effects of EMD are visible in PC2. There is a heterogenicity caused by one data point on THP1 controls.\u003c/p\u003e\u003cp\u003e\u003cem\u003eVolcano analysis of gene expression changes by EMD-exposed THP-1 and U937 cells.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTo understand the direction and magnitude of gene expression changes a Volcano plot was prepared (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The volcano plot represents a minimum 2.0-log10 significance and a 1.5-log2 fold- expression change. Based on these criteria, we recognized 5 and 30 upregulated genes in THP-1 and U937 cells, respectively. Also, 37 and 23 down-regulated genes were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In the THP-1 cells, the most obvious was the increased expression of S100A8, S100A9, and CD38, as well as the decrease in GINS4, GEM, RRM2, CAV1, ADM, CD48, and PDGFB. In the U937 cells, ITAG1, ITGA2, ITAG6, OLR1, and FN1 were apparently increased, while PDGFB, besides CCL1 and CCL8, were among the decreased genes. The volcano plot further highlights the differential response of the THP1 and U937 cells to EMD treatment and details are depicted in Supplement Files 4.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eHeat map of gene expression changes by EMD-exposed THP-1 and U937 cells\u003c/h2\u003e\u003cp\u003eNext, we showed the heat map of the 5/37 (42) and 30/23 (53) genes selected by the indicated thresholds in THP-1 and U937 cells. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e highlights the variance and magnitude of the differential gene expression. The heat map further confirms the heterogeneity within the cell response of THP-1 compared to U937 cells when exposed to EMD. Moreover, consistent with the PCA, one sample of the THP1 down-regulated genes resembles an outlier, and the data should be interpreted accordingly. The underlying data are summarized in Supplement Files 5.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eVenn analysis of genes regulated by EMD-exposed THP1 and U937 cells\u003c/h3\u003e\n\u003cp\u003eNext, a Venn analysis was performed on the differential response of THP-1 and U937 cells to EMD stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Supplement File 6). Venn analysis identified only two commonly regulated genes, with PDGFB being decreased in both THP-1 and U937 cells, whereas RIPOR2 was increased and decreased in the respective cell lines. This analysis further demonstrates the heterogeneity of how THP-1 and U937 cells respond to EMD.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThis analysis is restricted to the genes selected under a 1.5 log2 fold-change and a significance of 2.0 log10. Among the 42 and 53 significantly regulated genes in THP-1 and U937 cells, they have only two genes in common that EMD regulates.\u003c/p\u003e\u003cp\u003e\u003cem\u003eSTRING analysis of expression changes in THP-1 and U937 cells exposed to EMD.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe STRING analysis was performed to identify potential clusters of gene expression changes and details are summarized in Supplement File 7. THP1 cells showed eight clusters, highlighting \u0026ldquo;Regulation of neutrophil degranulation\u0026rdquo; (CD38, CD48, SLC12A8, SYK, ITGAM, C3AR1) and \u0026ldquo;Neutrophil aggregation\u0026rdquo; (S100A8, S100A9). In the U937 cells, a significant cluster was related to \"ECM-receptor interaction\u0026rdquo; (ITGA1, ITGA2, ITGA6, MMP3, MMP7, MMP25, IL7R, IL21R, FAP, SPP1, FN1, PROCR, and PDGFB) and \u0026ldquo;Monocyte chemotaxis\u0026rdquo; (CCL1, CCL20, CCL4L2). (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe protein\u0026ndash;protein association network and functional enrichment analyses of THP-1 \u003cem\u003e(A)\u003c/em\u003e and U937 \u003cem\u003e(B)\u003c/em\u003e cells, based on differentially expressed genes 42 and 53, are depicted.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eG: Profiler analysis of gene expression changes in THP-1 and U937 cells by EMD\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFollowing was a functional enrichment analysis of the 42 genes that were strongly regulated (5 up-regulated and 37 down-regulated genes) in THP-1 cells (Figure 6). Gene ontology confirmed the expected enrichment for Toll-like receptor binding (GO:0035325) linking S100A8, S100A9, and SYK. Enrichment was also achieved for leukocyte migration (GO:0050900) with S100A8, S100A9, PDGFB, SYK, SPNS2, APOD, CYP19A1, EDN1, HSD3B7, C3AR1, JAM2 and RIPOR2. As shown in Figure 7 and in Supplement Files 8, the 53 regulated genes in U937 cells exhibited enrichment for chemokine activity (GO:0008009), specifically with CCL1, CCL8, CCL4L2, and CCL20. Also highlighted is collagen binding (GO:0005518) with ITGA1, ITGA2, FN1 and PDGFB. Moreover, we have collagen catabolic process (GO:0030574) including MMP3, MMP7, MMP25, FAP and integrin complex (GO:0008305) with ITGA1, ITGA2 and ITGA6.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunctional enrichment analysis (over-representation analysis (ORA) or gene set enrichment analysis) was performed using the g: Profiler online tool. The top significant pathways were highlighted and labeled numerically. The p-value was adjusted (Padj) for multiple testing using the Benjamini-Hochberg method.\u003c/p\u003e\n\u003cp\u003eFunctional enrichment analysis (over-representation analysis (ORA) or gene set enrichment analysis) was performed using the g:Profiler online tool. The top significant pathways were highlighted and labeled numerically. Using the Benjamini-Hochberg method, the P value was adjusted (Padj) for multiple tests.\u003cbr\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eRT-PCR analysis of selected genes in THP-1 and U937 cells exposed to EMD and SB431542\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA RT-PCR analysis of selected genes was used to confirm the RNAseq analysis and to support the notion that cell responses to EMD are linked to TGF-\u0026beta; activity. EMD exposure of THP-1 and U937 cells caused the expected increase in S100A8, S100A9, CD38, and ITGA2, ITGA6, and OLR1 expression, respectively; however, not in the presence of the TGF-\u0026beta; receptor type I kinase-inhibitor SB431542 (Figure 8 and Supplement Files 8). The exception was that the decrease of PDGFB expression could not be reversed by SB431542, suggesting an expression change independent of TGF-\u0026beta; signaling (Figure 9). When freshly prepared primary CD14+ monocytes were exposed to EMD, CCL20 increased at least 3-fold; however, no significant changes were noticed for S100A8, S100A9, CD38, ITGA2, ITGA6, and OLR1 (data not shown).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eEMD is a tool used to facilitate periodontal and peri-implant regeneration, inspiring preclinical research on translating clinical endpoints into cellular responses [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. EMD is initially derived from the tooth germs of piglets and thus represents a broad spectrum of proteins and possibly other bioactive components; a complex cellular response can be expected. Accumulating evidence suggests that, apart from mesenchymal cells [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], hematopoietic cells are also targeted by EMD, but this has not been tested on THP-1 and U937 cells. Apart from the functional in vitro bioassays related to the differentiation of hematopoietic cells towards macrophages [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and osteoclasts [\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], we lack bioassays to evaluate the expression of highly regulated genes in response to EMD. These genes reflect the functional response of macrophages to EMD and may further support the estimation of the biological activity of the various batches of EMD in the production process. Thus, our overall motivation was to employ an RNA-seq approach to identify a panel of genes that are ideally strongly regulated by EMD in established THP1 and U937 cell lines, with a potential clinically relevant function in periodontal and peri-implant regeneration.\u003c/p\u003e\u003cp\u003eOne main finding of the present research was that only five genes were upregulated in THP1 cells, considering the respective threshold levels. The upregulated genes in THP-1 cells were S100A8, S100A9, CD38 and PIK3IP1 [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], RIPOR2. These findings align with those of HSC-3 carcinoma cells exposed to EMD, which upregulate S100A8 and S100A9 [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], both members of the S100 calcium-binding protein family. These proteins are commonly expressed in macrophages, where they exert their proinflammatory and immunosuppressive properties [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. S100A8 and S100A9 in saliva and gingival crevicular fluid have been proposed for screening established periodontitis [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Inflammatory mediators, such as TNF-α, increase their production in THP-1 cells [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Single-cell analysis revealed a macrophage subset expressing high levels of S100A8 and S100A9 in early gastric cancer tissues [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. In addition, CD38, a multifunctional transmembrane glycoprotein, is also increased in THP-1 cells, for instance by IFN-γ[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], erythritol [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], and retinoic acid, a vitamin A metabolite [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Daratumumab and Isatuximab, both antibodies binding to CD38, were granted therapy drug status for multiple myeloma [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Considering the clinical relevance and the robust increase by EMD, the expression of S100A8, S100A9, and CD38 in THP1 cells is one of the proposed bioassay markers.\u003c/p\u003e\u003cp\u003eAnother key finding was that EMD caused 37 genes to be down-regulated in THP-1 cells. Among the downregulated genes is PDGFB, which has also been observed in U937 cells. Support for this finding comes from observations that IFN-γ treatment of THP-1 cells caused the decreased expression of PDGFB [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Moreover, macrophage-derived PDGFB induces smooth muscle cell expansion, which may exceed pathological conditions, such as in pulmonary hypertension [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] and obesity-associated metabolic stresses, as it plays a significant role in regulating pericyte behavior during angiogenesis [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. However, the protective role of macrophage-derived PDGF-BB on nucleus pulposus cells was also shown [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Down-regulated genes further include MMP1, which is usually not expressed in THP1 cells but is induced by PMA stimulation [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. THP-1 expression of MMP-1 was associated with aldosterone-induced macrophage infiltration [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Significantly higher MMP-1 expression levels were found in the inflamed gingiva compared with healthy biopsies, and Filifactor blockade caused an upregulation of MMP-1 in THP-1 cells [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], suggesting a potential bioassay where EMD can be tested for modulation of high MMP-1 levels. Additionally, the levels of MMP-1 were higher at sites with periodontitis and peri-implantitis compared to sites with healthy teeth and implants [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. However, there are further down-regulated genes that need attention.\u003c/p\u003e\u003cp\u003eEMD also diminishes AMD in THP-1 cells, which are known to be producers of ADM [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. However, the secretion of TNF-α in LPS-stimulated THP-1 cells was reduced in the presence of recombinant AMD [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Thus, AMD is considered a multifaceted peptide implicated in vasodilation, hormone secretion, antimicrobial defense, cellular growth, angiogenesis, and, importantly, chronic pain [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. It was also identified in human gingival crevicular fluid [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Most strongly decreased by EMD is GINS4, a promoter of the cell cycle, but little is known about its impact on ferroptosis [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Moreover, partial loss-of-function mutations in GINS4 lead to NK cell deficiency with neutropenia [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Considering THP-1 is a tumor cell, the lowering of GINS4 by EMD may also serve as a bioassay in oncology; however, its clinical implication should be interpreted carefully. In addition, CAV1 is increasingly expressed in THP-1 cells by agonists of peroxisome proliferator-activated receptor gamma activation [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e], and CAV1 expression is lower in leukemia cancer cell lines, including THP-1, when compared with normal white blood cells [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. CAV1 partially mediates the role of gingipanins to permeabilize the blood-brain barrier [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e], and CAV1 knockout mice exhibit increased bone size and stiffness. Strong CAV1 immunoreactivity was detected in gingival epithelial and endothelial cells [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. Although a series of downregulated genes was identified, our THP-1 data revealed a larger variation; therefore, further refinement is needed in the selection of downregulated genes driven by EMD.\u003c/p\u003e\u003cp\u003eIn the U937 cells, 30 upregulated genes by EMD reached the threshold. Most impressively, and by the cluster analysis, is the significant increase in the expression of integrins: ITGA1, ITGA2, and ITGA6 by EMD. These transmembrane α integrins dimerize with β integrins, together serving as regulators of cell\u0026ndash;matrix and cell-cell interaction. For example, α1β1 and α2β1 were identified as collagen receptors [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. ITGA6 is effective in laminin binding. Integrin binding is not passive as it promotes outside-in signaling, thus affecting cell behavior [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. ITGA2 and FN1 are ERK-dependent targets of TGF-β signaling [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. The present data thus suggest that EMD-exposed U937 cells preferentially adhere to collagen- and laminin-rich ECM. This observation may lead to the establishment of an in vitro bioassay for cell adhesion to coated surfaces or biomaterials. Clinically, an increased expression of integrins in macrophages has potential relevance, for instance, in facilitating migration into a defect site and enabling local adhesion to the ECM [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. Likewise, lectin-like oxidized low-density lipoprotein receptor 1, encoded by OLR1, is enriched in a proinflammatory subset of macrophages [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e], together with CD36 [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. Functional assays may, therefore, test the impact of EMD on macrophage binding and internalization of oxLDL. Besides, IL7R should be mentioned in the context of macrophage infiltration under inflammatory conditions [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e], and MMP3 degrades various collagen types and other components of the ECM and activates other MMPs [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. CCL20, which is primarily chemotactic for lymphocytes, was increased in macrophages following aortic dissection [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e] and upon the polarization of THP1 cells [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e] and U937 cells [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. Our preliminary experiments with EMD-exposed CD14\u0026thinsp;+\u0026thinsp;blood-derived monocytes consistently showed a robust increase in CCL20 expression, further supporting our attempt to initiate future research on RNA-seq with primary monocytes.\u003c/p\u003e\u003cp\u003eU937 cells responded by 23 downregulated genes by EMD. For instance, EMD decreased the expression of the proteases MMP7 and MMP25. MMP7 is effective in cleaving casein, various collagen types, fibronectin, and proteoglycans, and MMP25 belongs to the membrane-type MMP (MT-MMP) subfamily [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. It is required for a proper innate immune response, such as to bacterial LPS, hypergammaglobulinemia, and reduced secretion of proinflammatory molecules [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]. Additionally, IL21R is required for infiltrating macrophages following infection [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e], and it also reduces the LPS-mediated secretion of inflammatory cytokines in macrophages [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. Concerning the decrease of the chemokines, CCL1 is involved in atherosclerosis [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e] and fibrosis [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e]. CCL8 was upregulated in the periodontal ligament during the initial stage of OTM [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e] and has been shown to impair muscle regeneration [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e]. Macrophages with high CCL4L2 expression were identified in response to pathogenic bacteria in the intestine [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e] and rheumatoid arthritis [\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e]. For review of chemokines in dentistry, see [\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e]. Moreover, potentially relevant for establishing a bioassay are SIGLEC1, an endocytic receptor that mediates clathrin-dependent endocytosis [\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e], and LILRB2, a class I MHC antigen receptor [\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e]. F2RL3, a receptor for activated thrombin or trypsin coupled to G proteins, is interesting [\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e], and CYSLTR1, a receptor for cysteinyl leukotrienes [\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e]. Apart from PDGFB, the selection of the gene set for a bioassay depends on the research question.\u003c/p\u003e\u003cp\u003eWe have recently exposed PBMCs to EMD in THP-1 cells [\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e]. Only FN1 and SERPINA1 are commonly upregulated genes, and PNOC is downregulated. In U937 cells, however, we have noticed that EMD also upregulates ITGA1, FN1, OLR1, SE-MA3C, SLC11A1, SNX10, TMEM52B, ALCAM, MS4A7, MS4A14, NMB, MRC2, and lowers MMP25, GDF15, SQLE, CD300A, and PTGIR \u0026ndash; similar to the effects observed in U937 cells. However, PBMCs represent cells, including the monocyte phenotype, with CD14\u0026thinsp;+\u0026thinsp;staining, while THP-1 and U937, due to their pre-differentiation, should be considered as macrophages. Interestingly, in PBMCs, EMD upregulates PDGFB [\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e]. However, PBMCs are a heterogeneous cell population, and we cannot differentiate between the cell types \u0026ndash; whether the overlapping expression changes caused by EMD are caused by monocytes, granulocytes, or lymphocytes. We are currently exploring this aspect using a single-cell RNA-seq approach, and our preliminary data have at least identified OLR1 and PDGFB as being increasingly expressed in the monocyte fraction of whole blood exposed to EMD, respectively. Thus, our data suggest that the U937 cell line is preferable for reflecting the response of primary blood cells.\u003c/p\u003e\u003cp\u003eThere is also a debate about whether the activity of EMD is related to amelogenins, as they play a role in tooth development. Evidence for the role of amelogenin comes from knockout mice, which show a progressive deterioration of cementum linked to increased osteoclastogenesis [\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e]. Moreover, mice lacking ameloblastin experience severe enamel hypoplasia [\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e]. These genetically modified mouse models elegantly underscore the critical importance of amelogenin and ameloblastin during tooth development; however, insights into tooth development should not be extrapolated to their function after tooth eruption, and evidence for the clinical activity of EMD is, if at all, indirect. New insights may derive from generating inducible amelogenin and ameloblastin transgenic mouse models, which could reveal the role of amelogenins after tooth eruption. This model may simulate the local application of EMD. Today, we know that molars from M180 amelogenin transgenic mouse lines are similar to those of wild-type teeth [\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e]. Future in vitro research should compare the cell response to EMD with recombinant amelogenin and ameloblastin, as well as TGF-β \u0026ndash; potentially using single cell RNAseq approach of whole blood or simply a bulk RNAseq of U937 or CD14\u0026thinsp;+\u0026thinsp;monocytes, and, also relevant, oral fibroblasts \u0026ndash; to identify to which extend the gene regulation caused by EMD is a consequence of amelogenesis and TGF-β activity, respectively.\u003c/p\u003e\u003cp\u003eFinally, we have selected a panel of genes for THP-1 and U937, not only to conform to some of the RNA-seq data, but, perhaps more importantly, to gain first insights into the role of TGF-β signaling in the cell lines that mediates the activity of EMD. In support of previous studies in mesenchymal and murine hematopoietic cells, SB431542 significantly blocked the effect of EMD in raising the expression of S100A8 in THP-1 and ITGAs in U937 cells. Nevertheless, when focusing on the decreased expression of PDGFB, SB431542 failed to block the EMD effects significantly. Therefore, it is worthwhile to conduct future studies to determine the extent to which SB431542 blocks the spectrum of EMD-regulated genes and to explore whether other bioactive components in EMD may contribute to the changes in PDGFB and possibly other genes. Additionally, there is a demand for studies comparing primary human monocytes or macrophages with the THP-1 and U937 cell responses, with the overall aim of narrowing down the panel of genes that represent primary cells while taking advantage of a reproducible bioassay situation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003eAuthor Contributions\u003c/em\u003e: Conceptualization, L.P. and R.G.; data curation, L.P.; formal analysis, L.P. and R.G.; methodology, L.P.; project administration, L.P. and X.H.; supervision, L.P. and R.G.; validation, L.P.; visualization, L.P.; writing\u0026mdash;original draft preparation, L.P. and R.G.; writing\u0026mdash;review and editing, L.P., X.H., and R.G. All authors have read and agreed to the published version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFunding\u003c/em\u003e: The University Clinic of Dentistry has supported this study. L.P. and R.G. received funds from the ITI Projects 1729-2023 and 1709-2022.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eData Availability\u003c/em\u003e: The datasets generated during the current study are available in the Gene Expression Omnibus (GEO) repository, under the accession numbers GSE307844. Differentially genes with an adjusted p-value of 0.05 are shown in Supplement File 1. All other data to generate the Figures are attached as Supplement Files 2-8.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAcknowledgments\u003c/em\u003e: We thank the Core Facility Genomics of the Medical University of Vienna for performing the RNA-seq analysis, and especially Sophia Derdak for her valuable support. EMD was a kind gift from Straumann \u0026Ouml;sterreich (Vienna, Austria).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConflicts of Interest\u003c/em\u003e: The authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate: The study was conducted by the principles outlined in the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003eConsent for publication: Informed consent was obtained from all participants in this study.\u003c/p\u003e\n\u003cp\u003eCompeting interests: The authors declare no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKinane DF, Stathopoulou PG, Papapanou PN. Periodontal diseases. 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J Biol Chem. 2003;278:35743\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1074/jbc.M306284200\u003c/span\u003e\u003cspan address=\"10.1074/jbc.M306284200\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-oral-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ohea","sideBox":"Learn more about [BMC Oral Health](http://bmcoralhealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ohea/default.aspx","title":"BMC Oral Health","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7240030/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7240030/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjectives: \u003c/strong\u003eEnamel matrix derivatives (EMDs) are applied in periodontal defects and gingival recession, a process where macrophages contribute to the clinical outcome. There is a need for standardized bioassays to better understand and monitor how EMD affects macrophages in vitro.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and methods: \u003c/strong\u003eWe propose using THP-1 and U937, both widely established monocytic cell lines, as bioassays in EMD research. Both cell lines have different origins, as THP-1 is a leukemia cell line, and U937 originates from the pleural effusion of a patient with histiocytic lymphoma. To understand their differential response to EMD, we employed an RNA-seq approach revealing changes in the genetic signatures of THP-1 and U937 cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eWhen applying a threshold of 1.5 log2 fold-change and a significance of 2.0-log10, we could identify 5/37 and 30/23 up- and down-regulated genes in THP1 and U937 cells, respectively. In THP-1, the upregulated genes included S100A8, S100A9 and CD38; downregulated gene included ADM, CD48, IL24, MMP1, and PDGFB. In U937, most striking was the increase of alpha subunit integrins ITGA1, ITGA2, ITGA6, and the decrease of genes including OLR1, CCL1, CCL4L2, CCL8, IL21R, MMP7, PDGFB and MMP25. We further show that the TGF-β receptor type I kinase inhibitor SB431542 blocked the expression changes of S100A8, S100A9, CD38, ITGA2, ITGA6, and OLR1 but failed to reverse PDGFB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThese data serve as a primer for developing macrophage bioassays to measure EMD activity in the context of TGF-β signaling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical relevance\u003c/strong\u003e: To identify a panel of genes, ideally being strongly regulated by EMD, in established THP1 and U937 cell lines, with a potential clinically relevant function in periodontal and peri-implant regeneration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number:\u003c/strong\u003e Not applicable.\u003c/p\u003e","manuscriptTitle":"RNA-seq of THP-1 and U937 exposed to enamel matrix derivative","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-22 19:23:39","doi":"10.21203/rs.3.rs-7240030/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"299669028006225129094470934908866680884","date":"2025-10-21T14:07:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-09T07:30:40+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-15T14:50:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-15T14:43:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-12T11:23:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Oral Health","date":"2025-09-12T09:37:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-oral-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ohea","sideBox":"Learn more about [BMC Oral Health](http://bmcoralhealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ohea/default.aspx","title":"BMC Oral Health","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b717a80d-5be4-402e-82a1-b972bd4c5aa0","owner":[],"postedDate":"October 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-10-22T19:23:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-22 19:23:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7240030","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7240030","identity":"rs-7240030","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}