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Transcriptome Profiling of IL-17A Preactivated Mesenchymal Stem Cells: A Comparative Study to Unmodified and IFN-[gamma] Modified Mesenchymal Stem Cells.

1. Introduction

Human bone marrow derived mesenchymal stem cells (MSC) pretreated with interleukin-17A (IL-17A) represent a novel immunomodulatory strategy and an alternative to interferongamma (IFN-[gamma]) treatment of MSC in enhancing MSC immunosuppression of T cells [1]. We have previously demonstrated that human MSC-17 potently suppresses human T cell proliferation and activation. In cocultures of MSC with purified human [CD4.sup.+][CD25.sup.-] T cells, MSC-17 induced high numbers of functionally suppressive iTregs [1]. Whilst MSC17 appeared to be superior modulators of T cells, mechanisms exclusive to MSC-17 mediated immunomodulation warrant further investigation.

IL-17A is a member of the family of IL-17 cytokines secreted predominantly by the T helper 17 (Th17) subset of [CD4.sup.+] T cells. IL-17A is a potent proinflammatory mediator and is involved in the pathogenesis of autoimmune diseases, allergic responses, and other immune cell mediated diseases including allograft rejection, sepsis, and graft versus host disease (GvHD) [2, 3]. Apart from the pathogenic roles of IL-17A, this cytokine is important for host defense response against fungal and bacterial infections [3, 4]. The IL-17A homodimer signals through the IL-17RA and IL17RC dimeric receptor complex, where binding of IL-17A homodimer to the IL-17RA/RC complex recruits the key cytosolic adaptor molecule Act1 (NF-kappa B-activating protein), that is known to be the master mediator of downstream IL-17 signaling [3, 5]. Act1 binds to the IL17RA/RC complex via its SEFIR (SEF/IL-17R) domains and this complex then recruits TRAF6 (TNF receptor-associated factor 6), leading to the activation of several downstream signaling pathways including the MAPKs-AP-1 (mitogen-activated protein kinases, MAPKs; activator protein-1, AP1), C/EBPs (CCAAT/enhancer-binding proteins) and NF[kappa]B (nuclear factor kappa B). Activation of these signaling cascades induces the gene expression of antimicrobial peptides, chemokines, MMPs, and proinflammatory cytokines as shown in other cell types such as endothelial cells, epithelial cells, and fibroblasts [3, 4]. IL-17A has emerged to be a growth factor for MSC by activating the Akt-Erk-MEK-p38 transduction molecules involved in MAPK signaling cascades [6-8]. Published work from our laboratory, demonstrated for the first time that IL-17A also enhances the immunomodulatory capacity of human MSC [1].

IFN-[gamma] is produced predominantly by [CD8.sup.+] T cells and NK cells and at lower levels by [CD4.sup.+] T cells [9]. IFN-[gamma] binds to a heterodimeric cell surface receptor complex consisting of the interferon-gamma receptor 1 (IFNGR1) and IFGR2, activating the classical JAK-STAT (signal transducer and activator of transcription) signaling pathways [10]. Activation of this pathway regulates several downstream cascades and induces expression of many genes, thereby contributing to the diverse biological effects of IFN-[gamma] in different cell types [10-12]. IFN-[gamma] activates macrophages to induce antitumor [13] and antimicrobial activities [14]. It is also well established that IFN-[gamma] induces antigen processing and presentation pathways in different cell types for MHC antigen presentation to T cells [9, 15-17]. In B cells, IFN-[gamma] regulates immunoglobulin production and class switching [16, 18]. IFN-[gamma] also attracts leukocytes and favours the growth, differentiation, and maturation of many cells types [11, 16]. IFN-[gamma] is classically known as a cytokine that favours Th1 cell development [16, 19]. In an allotransplantation setting, IFN-[gamma] promotes antigen-specific Th1 differentiation that drives cell mediated allograft rejection [20]. Together, these findings suggest the potent proinflammatory role of IFN-[gamma].

The role of IFN-[gamma] in MSC immunomodulation, reparative properties, and homing potential has been extensively reviewed as previously published [21]. IFN-[gamma] treated MSC (MSC-[gamma]) have enhanced immunomodulatory properties but are potentially immunogenic when administered in allogeneic or third-party hosts [1]. In this study, microarray and bioinformatics approaches were used to further identify novel candidate molecules expressed by MSC-[gamma] and MSC-17 that enhance the immunomodulatory properties of MSC. Genes and biological processes that may contribute to MSC-[gamma] immunogenicity in allogeneic or third-party hosts were also explored.

2. Materials and Methods

2.1. MSC Culture and Characterisation. Human bone marrow aspirates were obtained from the posterior iliac crest of normal adults volunteers (subjects with informed consent; age 20-35 yr) according to guidelines approved by the Human Ethics Committee of the Royal Adelaide Hospital, Australia (Protocol 940911a). Bone marrow derived MSC cultures were established and maintained as previously described [22, 23]. Cryopreserved MSC were cultured to log-phase and used at passage 6 in experiments. The immunophenotype of culture expanded MSC and their ability to differentiate into adipocytes, osteocytes, or chondrocytes have been confirmed and published [1].

2.2. Cytokine Treatment of MSC. MSC were seeded in tissue culture flasks at a density of 4000 cells/[cm.sup.2] and were allowed to adhere overnight. Fresh MSC media containing either no cytokines or recombinant human cytokines, 500 U/ml IFN-[gamma] (eBioscience) or 50 ng/ml IL-17A (Peprotech), were added to the MSC cultures to derive UT-MSC, MSC-[gamma], or MSC17, respectively. At day 5, cytokines were washed out with Hank's Balanced Salt Solution (HBSS, Sigma) and modified MSC were used for microarray gene expression profiling and analysis.

2.3. Human MSC RNA Isolation. MSC were harvested using 0.25% trypsin/EDTA (Sigma) for 4 min, 37[degrees]C, and rinsed with 5% FBS/HBSS and RNA was extracted according to the protocol established by the Adelaide Microarray Centre ( Briefly, total RNA was extracted by dissolving the cell pellet in 500 [micro]L TRIzol reagent (Invitrogen) and 100 [micro]L chloroform was added to the mixture. The mixture was kept on ice for 15 min followed by centrifugation at 6500 x g for 30 min, 4[degrees]C. The upper aqueous phase was retained and mixed with an equal volume of 70% ethanol in diethylpyrocarbonate [H.sub.2]O. Total RNA was further purified using the RNeasy mini kit (Qiagen) with the following modification: DNA was digested using the DNase I from the RNase-free DNase set (Qiagen). The quantity of total RNA was measured using NanoDrop 1000 (Thermo Scientific). Samples were adjusted to a concentration of 100 ng/[micro]L for microarray and were sent to the Adelaide Microarray Centre, University of Adelaide, for microarray gene expression profiling. The RNA sample was determined using the Agilent RNA Bioanalyzer. Only RNA samples with RNA integrity number (RIN) of [greater than or equal to] 8 were used for microarray analysis.

2.4. Microarray Analysis. RNA extracted from human MSC samples were analysed using the Affymetrix Human Gene 2.0 ST Array (Affymetrix Inc., High Wycombe, UK) for gene expression profiling. Microarray gene expression profiling was performed on UT-MSC, MSC-[gamma] and MSC-17 from 3 human MSC donor biological replicates (passage 6). Microarray experiments were conducted by the Adelaide Microarray Centre, University of Adelaide.

2.5. Microarray Quality Control and Gene Expression Analysis. Probe cell intensity (CEL) files were obtained from the Adelaide Microarray Centre. The Expression Console Software (Affymetrix) was used for data quality control, normalization, and differential gene level analysis. CEL files of each array showed no major issues or damage with the images. No outlier samples were identified based on the configurable QA/QC metrics. The RMA (robust multiarray analysis) algorithm was used to perform background subtraction, normalization, and summarization of probe sets. CHP files were generated from the Expression Console Software for further Principal Component Analysis (PCA) and gene level summarization using the Transcriptome Analysis Console (TAC) software (Affymetrix). After normalization, UT-MSC, MSC-[gamma], and MSC-17 from 3 donor samples of each treatment group were averaged and an unpaired one-way ANOVA was performed with significantly regulated genes identified by p < 0.05 and fold changes < -2 and >2. Gene lists for comparison of MSC-17 versus UT-MSC, MSC-[gamma] versus UT-MSC, and MSC-17 versus MSC-[gamma] were generated for subsequent bioinformatics analysis.

2.6. Functional Enrichment Analysis by DAVID. Gene lists for comparison of MSC-17 versus UT-MSC, MSC-[gamma] versus UT-MSC, and MSC-17 versus MSC-[gamma] were analysed for their biological functions using the Database for Annotation, Visualisation and Integrated Discovery (DAVID; The gene list was uploaded using the official gene symbol onto DAVID for functional annotation clustering analysis with medium classification stringency, enrichment scores > 1.5, and p < 0.05 [24]. Functional annotation clustering analysis based on DAVID's default settings was performed. The gene sets were also subcategorised based on functional annotation of interest such as biological process (GOTERM_BP_FAT), molecular function (GOTERM_MF_FAT), and cellular component (GOTERM_CC_FAT).

2.7. Real-Time PCR Gene Validation. Genes of interest identified by microarray were validated by real-time PCR (RT-PCR) as previously described [1]. Gene specific human Taqman[R] primers MMP1 (Hs00899659_m1), MMP13 (Hs00233992_ml), CCL2 (Hs00234140_m1) CCL8 (Hs04187715_m1), CXCL6 (Hs00605742_m1), C3 (Hs00163811_ml), CH25H (Hs02379634_s1), and LBP (Hs01084621_ml) (Applied Biosystems) were used for gene expression analysis. Samples were run in triplicate and data were presented and normalized to the housekeeping gene hypoxanthine phosphoribosyltransferase-1 (HPRT1) (Hs99999909_ml). Mean normalized expression was calculated using the Qgene Module software as previously described [25].

3. Results

3.1. Transcriptome Profiling of UT-MSC, MSC-[gamma], and MSC17. The transcriptome differences between UT-MSC, MSC-[gamma], and MSC-17 from 3 different human MSC donors were compared in this study. Principal Component Analysis (PCA) was performed to visualise variances between the 3 donors and treatment groups. PCA analysis revealed that the 3 donor replicates of MSC-[gamma] "clustered" together. The gene expression pattern in the MSC-[gamma] groups were clearly distinct from UTMSC and MSC-17 (Figure 1). Microarray analysis revealed that 1278 genes (902 upregulated; 376 downregulated) were differentially regulated between MSC-[gamma] and UT-MSC. The top 30 upregulated and downregulated genes in the MSC-[gamma] were shown in Table 1.

There were however donor variances that exist between MSC-17 and UT-MSC. Among the 3 MSC donor samples evaluated, 2 MSC donors (i.e., donor C and F) "clustered" together and were distinct from UT-MSC (Figure 1). It should also be noted that in donor C and F MSC-17 "clusters," there was less variability in the gene expression profile in MSC-17 versus UT-MSC compared to the MSC-[gamma] versus UT-MSC groups. Donor M on the contrary had a different gene expression pattern in both UT-MSC and MSC-17. This clustering analysis in general supports a lesser degree of change in the gene expression profile of MSC with IL-17A than IFN-[gamma]. Based on these 3 MSC donors, microarray analysis identified that only 67 genes (39 upregulated; 28 downregulated) were differentially regulated between MSC-17 and UT-MSC (Table 2).

The gene expression profile of MSC-17 versus MSC-[gamma] was also evaluated. The clustering of the 3 MSC donors in the MSC-17 and MSC-[gamma] comparison groups was more distinct (Figure 1) when compared to MSC-17 and UT-MSC. Microarray analysis revealed that 1806 genes (391 upregulated; 1415 downregulated) were differentially regulated between MSC-17 and MSC-[gamma]. The top 30 upregulated and downregulated genes in the MSC-17 versus MSC-[gamma] comparison group were shown in Table 3. Volcano plots (Figure 2) and supervised hierarchical clustering of the differentially expressed genes (Figure 3) provided a global visualisation of genes regulated by IL-17 or IFN-[gamma] treatment of MSC compared to UT-MSC.

3.2. MSC-[gamma] Enriched for Genes Associated with Increased Immunogenicity. Upregulated and downregulated gene lists were submitted to DAVID for functional annotation clustering analysis to identify gene sets that were enriched in MSC-[gamma]. There were 90 and 62 official gene symbols from the upregulated (see Table S1 in Supplementary Material available online at and downregulated (Table S2) gene entry lists, respectively, that were unmapped by DAVID. These were mainly noncoding genes including microRNA (miRNA), long noncoding RNA (lncRNA), and small nucleolar RNA (snoRNA). Gene ontology analysis by DAVID functional annotation clustering was performed on the upregulated and downregulated MSC-[gamma] versus UT-MSC gene lists to identify enriched gene sets for biological processes (Tables S3, S4), molecular functions (Tables S5, S6), and cellular components (Tables S7, S8).

Gene ontology analysis for biological processes of upregulated MSC-[gamma] genes (Table S3) uncovered highest enrichment of genes associated with antigen processing and presentation via MHC class I (annotation cluster 1, enrichment score 8.03). These genes were mainly HLA type genes and have roles in antigen presentation. Enriched genes in annotation cluster 1 also include aminopeptidases that hydrolyse antigenic peptides for MHC class I peptide binding and antigen presentation (e.g., endoplasmic reticulum aminopeptidase ERAP1 and ERAP2), peptide transporter genes (e.g., transported associated with antigen processing, TAP2), and other genes involved in the antigen processing and presentation pathway (e.g., TAP binding protein, TAPBPL; [[beta].sub.2] microglobulin, B2M; CD74). Gene sets involved with antigen processing and presentation via MHC class II were also upregulated in the MSC-[gamma] groups (annotation cluster 4, enrichment score 4.45). In annotation cluster 2 (enrichment score 6.06) there were also enriched gene sets involved in immune response activation (innate, adaptive, and lymphocytes mediated immunity), humoral response (immunoglobulin mediated immune response, B cell mediated immunity, and humoral immune response mediated by circulating immunoglobulin), and complement pathways (classical and alternative) activation.

Apart from genes that are involved in increased MSC-[gamma] immunogenicity, there were genes with regulatory roles upregulated in MSC-[gamma] (Table S3). For example, these gene sets were involved in the regulation of programmed cell death, apoptosis, translation regulation, protein modification, transcription regulation and DNA binding activity, cell-cell communication, and signal transduction as well as the regulation of cytokine production. Moreover, genes upregulated in the MSC-[gamma] group were enriched for the TGF-[beta] receptor signaling pathway (annotation cluster 19, enrichment score 1.74, e.g., FMOD, CCL2, MAPK3K1, SMAD6, GDF15, and TGFB2). Other genes of interest upregulated in MSC-[gamma] include IL-6, toll-like receptor-3 (TLR3), TLR4, and indoleamine 2,3-dioxygenase (IDO), with the gene ontology term for positive regulation of defense response. There was also upregulation of the PD-L1 transcript in MSC-[gamma] compared to UT-MSC (3.46-fold, p < 0.0104; data not shown), consistent with the observed increase in cell surface protein expression of PD-L1 following IFN-[gamma] pretreatment of MSC, as we have previously published [1]. Regulatory genes with nucleotide binding activity and transcription (corepressor, repressor, and cofactor) activity were also enriched and upregulated in MSC-[gamma] as identified by DAVID gene ontology analysis for molecular function (Table S5).

MSC-[gamma] have enhanced migratory potential to sites of inflammation [21]. Based on DAVID analysis for biological processes, we have identified gene sets in annotation cluster 10 (enrichment score 2.78) that were enriched for the gene ontology terms regulation of cell motion, cell migration, and locomotion (Table S3). These upregulated MSC-[gamma] genes include chemokines (CXCL10, CXCL16), intracellular adhesion molecule-1 (ICAM1), IL-6, and VEGFA. The upregulation of chemotactic factors that may increase MSC-[gamma] homing potential was also identified when gene ontology analysis for molecular function was performed on DAVID. In annotation cluster 3 (enrichment score 3.10; Table S5), genes were enriched for chemokine receptor binding and chemokine activity. These chemokines include CCL13, CCL2, CXCL16, CXCL9, CCL8, CXCL11, and CXCL10.

Based on the downregulated MSC-[gamma] versus UT-MSC gene list, we identified that there were genes highly enriched for the gene ontology terms for biological processes involving extracellular matrix or structure organisation (annotation cluster 1, enrichment score 11.10; Table S4), consistent with our previous observation of changes in MSC-[gamma] morphology from fibroblastic-like appearance to a hypertrophic flattened irregular shape [1]. These were mainly collagen type genes (collagenases I, III, IV, V, XI, XII, and XIV). Interestingly, the downregulated gene sets also have enriched terms for biological processes involved in the cell division cycle (annotation cluster 2, enrichment score 8.80; Table S4) These downregulated genes were essential for M phase, nuclear division, mitosis, and cell division. Genes essential for regulation of cell-cycle division were also downregulated in MSC-[gamma] (annotation cluster 7, enrichment score 2.03), in coherence with the observation of decreased MSC-[gamma] growth kinetics compared to UT-MSC [1].

Gene ontology analysis for cellular components (Tables S7, S8) of the differentially regulated genes in the MSC-[gamma] versus UT-MSC groups uncovered that these genes were located in the extracellular space (or) region (annotation cluster 1, enrichment score 2.69, Table S7; enrichment score 12.76, Table S8). Many downregulated genes were located in collagen, the main structural protein in the extracellular region.

3.3. MSC-17 Enriched for Genes Associated with Chemotaxis. Differentially regulated genes were submitted to DAVID for functional annotation clustering to identify gene sets that were enriched in MSC-17. Genes that were mapped by DAVID were shown in Table 2. There were 23 genes from the gene entry list that were unmapped by DAVID (Table S9). These include noncoding genes, lncRNA, ribosomal RNA (rRNA), snoRNA, and miRNA.

Functional annotation clustering analysis was first performed using DAVID's default settings (Table S10) to identify overall gene sets that were highly enriched in MSC-17 compared to UT-MSC. Annotation cluster 1 with the highest enrichment score (3.58) had enriched terms for genes residing in the extracellular region, roles in inflammatory response, response to wounding, defense responses, signaling, and disulfide bonds (Table S10). Gene ontology analysis of biological processes also revealed that MSC-17 compared to MSC-[gamma] were upregulated and enriched for genes involved in angiogenesis (e.g., angiogenin, CXCL12, tissue plasminogen activator, and collagens), wound healing, and chemotaxis responses (Table S14). Interestingly, some gene sets were enriched for gene ontology terms such as glycosylation and glycoproteins (Table S10), which may relate to posttranslational modification processes [26, 27]. There was also high enrichment of genes involved in chromatin remodelling processes (enrichment score 7.35, Table S14), suggesting the potential gene expression regulatory roles of MSC-17.

Human MSC-17 were shown to be superior at regulating T cell inflammatory responses by suppressing T cell proliferation, activation, and secretion of proinflammatory cytokines [1]. In annotation cluster 3 (enrichment score 2.48, Table S10), genes such as IL-6, C3, serum amyloid A1 (SAA1), and lipopolysaccharide binding protein (LBP) were enriched for regulation of immune responses. IL-6, SAA1, and LBP also have roles in regulation of cytokine production.

Gene ontology analysis by DAVID functional annotation clustering was also performed on the MSC-17 versus UTMSC and MSC-17 versus MSC-[gamma] gene lists to specifically determine enriched gene sets for biological processes (Tables S11, S14, and S15), molecular functions (Tables S12, S16), and cellular components (Tables S13, S17, and S18) in MSC-17. There was no significant enrichment of gene sets for molecular functions in the downregulated gene list of MSC-17 versus MSC-[gamma] comparison group.

MSC-17 were previously shown to mediate Treg induction via cell-cell contact dependent mechanisms [1]. To identify potential cell surface candidate molecules that mediate MSC-17 induction of Tregs, the cellular compartments of genes enriched in the MSC-17 were also evaluated. Functional enrichment for biological processes identified a set of up-regulated genes (IL-6, CCL8, SLC22A3, STC1, and CXCL6) that were enriched for the gene ontology term cell-cell signaling (fold enrichment: 4.9; p < 0.0148; annotation cluster 2, Table S11). Chemokines CCL2, CCL8, and CXCL6 detected by DAVID's functional enrichment for molecular function (Table S12) showed evidence that these gene sets have a different range of binding potential including chemokine receptor, heparin, glycosaminoglycan, pattern, and polysaccharide binding activities. These MSC-17 enriched genes, mainly the chemokines and MMPs, were located in the extracellular space (or) region (Table S13).

Biological processes (GOTERM_BP_FAT; Tables S11, S14) and molecular functions (GOTERM_MF_FAT; Table S12) of MSC-17 enriched genes were mainly associated with cell migration and chemotaxis responses. MMPs were also highly enriched in the MSC-17 groups. Specifically, MMP13 (FC 15.6) and MMP1 (FC 2.4) were induced in the MSC-17 groups as detected by microarray gene expression analysis (Tables 2 and 3). DAVID's bioinformatics analysis revealed that these genes were enriched for gene ontology terms such as secreted, extracellular space, signal, disulfide bond, glycosylation, glycoproteins, and response to stimulus (Table S10). The MSC-17 versus UT-MSC gene list when analysed by DAVID functional annotation chart (default setting) showed that these MMPs where highly enriched for metal ion binding, peptidase, and collagen degradation functions (Table S10).

3.4. MSC-17 Express Chemokines and Matrix Metalloproteinases. To validate the microarray data of MSC-17, upregulated genes were evaluated for their gene expression by RTPCR (Figure 4). IL-17A induced the expression of MMP1, MMP13, CXCL6, C3, CH25H, and LBP in MSC as determined by microarray and validated by RT-PCR (p < 0.05). CCL2 and CCL8 were highly expressed in both MSC-[gamma] and MSC-17 compared to UT-MSC, consistent with the microarray data. CCL2 gene expression increased by 8.2- and 5.9-fold in MSC-[gamma] and MSC-17, respectively, relative to UT-MSC. CCL8 expression on the other hand was comparable between MSC-[gamma] and MSC-17. Although the gene expression levels varied between the 3 MSC donors, these genes were consistently upregulated relative to UT-MSC in all the MSC donors.

4. Discussion

IFN-[gamma] preactivation of human MSC induced the expression of various immunoregulatory molecules including IDO, TLR3/4, IL-6, and PD-L1 that may enhance the inhibitory activity of MSC-[gamma] to mediate T cell suppression. IDO is a well characterised immunosuppressive molecule expressed by MSC upon induction with IFN-[gamma] [1, 28-30]. Administration of IDO deficient MSC ([IDO.sup.-/-]MSC) or inhibition of IDO activity resulted in accelerated kidney allograft rejection, decreased intragraft, or circulating Tregs and showed absence of donor-specific tolerance [29]. [IDO.sup.-/-]MSC were also incapable of inhibiting donor DC maturation and function, thus enabling DC to stimulate strong recipient T cell proliferative responses [29]. Consistent with previous literature [28, 29, 31], gene expression analysis revealed that IDO was the most highly induced gene in MSC-[gamma] and may be the key candidate molecule bywhich MSC-[gamma] mediate enhanced T cell immunosuppression [1].

MSC constitutively express a range of TLRs, including TLR3 and TLR4 [32, 33]. Activation of TLR3 and (or) TLR4 amplifies MSC trophic factors, antimicrobial activity, and immunosuppressive potential, thereby enhancing MSC therapeutic potency [33-36]. Both TLR3 and TLR4 were upregulated in MSC-[gamma] compared to UT-MSC. Activation of TLR3 and TLR4 signaling with poly I:C or LPS, respectively, induced IDO expression in MSC [33]. TLR-driven induction of IDO in MSC resulted in the degradation of tryptophan and production of immunosuppressive kynurenines [33]. TLR3 activation has also been linked to expression of IL-6 in MSC [34]. IL-6 mediates the inhibitory effects of MSC on DC differentiation, maturation, and function [37, 38]. Consistent with our previous report, the upregulation of IL-6 transcripts in MSC-[gamma] and high protein concentrations of IL-6 in MSC-[gamma]-T cell coculture supernatants suggest that MSC-[gamma] secreted IL6 may be involved in suppression of proinflammatory T cell responses [1]. Nevertheless, TLR activation in MSC is known to abrogate their immunosuppressive properties [39, 40]. The effects of TLR signaling in MSC are still not fully understood and remain to be further investigated.

TLR3/4 preactivated MSC have enhanced leukocyte binding activity mediated by the induction of the adhesion molecule ICAM-1, consistent with upregulation of ICAM-1 in MSC-[gamma] [35]. ICAM-1 together with TLR3 and TLR4 were among the genes enriched for the gene ontology term positive regulation of immune system process, suggesting a potential biological role of TLR3/4 in MSC-[gamma] induction of ICAM-1 [35]. Additionally, the upregulation of chemokines such as CXCL9, CXCL10, CXCL11, CXCL16, CCL2, CCL8, and CCL13 detected by microarray may facilitate T cell recruitment to MSC-[gamma]. Mouse MSC preactivated with IFN-[gamma] and TNF-[alpha] induced CXCL9 and CXCL10 [41]. The production of these chemokines was abrogated by IFN-[gamma] neutralization [41]. Moreover, the blockade of CXCR3, a T cell receptor for chemokines CXCL9 and CXCL10, eliminated T cell chemotaxis towards MSC and subsequent MSC inhibition of T cell proliferation [41]. These studies concluded that cytokines induce MSC-expression of chemokines to drive T cell recruitment into close proximity with MSC, enabling MSC to suppress T cells through the secretion of immunosuppressive molecules [41, 42]. Chemokines also increased the in vivo migratory properties of human MSC-[gamma] to sites of inflammation in colitis mouse models [43]. Studies to validate the functional role of human MSC-[gamma] derived chemokines and ICAM-1 in the recruitment and subsequent modulation of T cell responses as well as in MSC-[gamma] homing to sites of inflammation in vivo are required. Hence, IFN-[gamma] directly induces an array of immunosuppressive molecules in MSC and may further amplify the secretion of other MSC-inhibitory molecules such as IDO, IL-6, and ICAM1 via TLR3/4 activation. MSC-[gamma] with higher proximity to leukocytes may serve as an additional mechanism by which MSC-[gamma] increase their modulatory activity on T cells.

Despite being highly immunosuppressive with enhanced homing and reparative capacities, allogeneic MSC-[gamma] are ineffective in vivo due to their increased immunogenicity [44-46]. A large number of genes upregulated in MSC-[gamma] were involved in the antigen processing and presentation pathways of MHC classes I and II. Antigen processing and presentation occurs via the cytosolic [47-51] or endocytic pathways [51, 52]. In the cytosolic pathway, degraded intracellular proteins are transported to the rough endoplasmic reticulum (RER) via TAP, a heterodimer consisting of TAP1 and TAP2. These peptides are further trimmed by aminopeptidases ERAP to enable optimal peptide loading onto MHC class I molecules. MHC class I components comprise the class I MHC [alpha]-chain and the B2M chain. This MHC class I molecule associates with the chaperone molecules tapasin, calreticulin, and ERp57. Tapasin (TAPBPL) recruits the MHC I molecules into proximity to TAP, allowing efficient peptide loading onto MHC class I molecules, subsequently stabilizing the peptideclass I molecule complex. The class I MHC-peptide complex is then transported to the plasma membrane for antigenpeptide presentation to [CD8.sup.+] T cells [47-51]. Induction of ERAP, TAP2, TAPBPL, and B2M, genes involved in this cytosolic pathway was evident in MSC-[gamma] and correlated with the observed upregulation of MHC class I in these cells [1]. We have also shown that MHC class II is induced in MSC-[gamma] [1]. In the endocytic pathway, assembly of MHC class II occurs in RER where the [alpha]- and [beta]-chain associate and this newly synthesised class II MHC complex binds to the invariant chain (Ii, CD74). As MHC class II-Ii complex is translocated into the endosomal compartment, the Ii chain is degraded, leaving the CLIP fragment (class II associated Ii peptide) bound to the MHC II peptide binding cleft. HLADM catalyses the exchange of CLIP with the antigenic peptide. The MHC class II peptide complex is then transported to the plasma membrane for antigen presentation to [CD4.sup.+] T cells [51, 52]. We detected high expression of CD74 and HLA-DM in MSC-[gamma], supporting the induction of MHC class II on these cells. Alloimmune responses against UT-MSC are mediated by the recognition of allogeneic MHC molecules by recipient [CD4.sup.+] and [CD8.sup.+] memory T cells [53]. MHC class II expression on allogeneic MSC is also known to induce alloimmune responses in cocultures with MHC-mismatched responder cells [54]. Therefore, the amplification of this antigen processing and presentation machinery suggests that MSC-[gamma] are highly immunogenic and can potently prime proinflammatory T cell responses in allogeneic hosts.

Moreover, MSC-[gamma] were enriched for gene sets involved in augmentation of the humoral immunity and complement pathways activation. Our data may explain previously published data, where MSC-[gamma] infused mice had higher levels of circulating anti-donor IgM and IgG alloantibodies, which resulted in the rapid induction of antibody-mediated rejection [44]. Although MSC-[gamma] lack the expression of costimulatory signals (CD80, CD83, and CD86) to function as APC to mediate direct T cell allorecognition and activation [1, 55-58], we speculate that MSC-[gamma] induce allogeneic T cell responses through the indirect or semidirect pathways of allorecognition [21, 59]. Allogeneic MHC-peptide transfer from MSC-[gamma] could be more rapid compared to UT-MSC due to high expression of MHC molecules. This enables allogeneic MHC-peptide to be recognised by recipient T cells through the semidirect pathway. Understanding mechanisms of MSC-[gamma] immunogenicity may enable the targeting of MSC through different pathways of activation to increase their immunomodulatory function whilst retaining MSC in a nonimmunogenic and inert state.

Human MSC-17 showed superior suppression of T cell responses and were able to induce Tregs with minimal immunogenicity [1]. MSC constitutively express a range of MMPs including MMP2, membrane type 1 MMP (MTIMMP), tissue inhibitor of MMP1 (TIMP1), and TIMP2 [60-62]. These MMPs are essential for MSC invasion and migration across the extracellular matrix (ECM) as demonstrated in in vitro transendothelial migration assays [62]. Absence of MMPs impairs MSC transmigration capacity across Matrigels [60-62]. In response to IL-1[beta] and TNF-[alpha], MSC have also been shown to amplify the expression of MMP2, MTI-MMP, and (or) MMP9 in MSC, thereby promoting MSC invasiveness across the basement membrane [60]. Here, we demonstrated that IL-17A induced the gene expression of MMP13 and MMP1 in MSC. These MMPs were highly enriched for collagen degradation and metabolic processes, suggesting that these factors may be essential for MSC-17 to invade the ECM.

MSC-derived MMPs also have proteolytic activity on chemokines [63, 64]. MMP processing of CC chemokines convert the biochemical properties of the chemokine target molecules from an agonist to an antagonist form with anti-inflammatory properties in vivo [65]. MSC-derived MMP1 cleaves CCL2, leading to the generation of CCL2 with suppressive properties on B cell production of immunoglobulins and in [CD4.sup.+] T cell activation [63, 64]. We showed that MMP1, CCL2, and MMP13 were upregulated in human MSC-17. Evaluating the functional role of MMP-processed chemokine derivatives in MSC-17 immunomodulation on T cells in this study remains to be elucidated.

MMP-2 and MMP-9 secreted by MSC are known to cleave and reduce CD25 expression on T cells, thus impairing T cell activation and proliferation [66]. Administration of MMP inhibitors in an islet allotransplant model abrogated the suppressive effect of MSC on alloreactive T cells, resulting in allograft rejection. This study concluded that MMPs are crucial for MSC immunosuppression [66]. We have previously shown that MSC-17 further downregulated CD25 expression on [CD4.sup.+] effector T cells compared to UT-MSC, a process partially mediated by cell contact dependent mechanisms [1]. The involvement of MSC-17-derived MMP13 and MMP1 in downregulating CD25 on T cells has not been previously established. Blocking MMP13 activity using specific inhibitors may provide insights on its role in inhibiting T cell activation.

Apart from the upregulation of chemotactic transcripts, MSC-17 were also enriched for genes involved in wound healing and angiogenesis. Tissue plasminogen activator (PLAT) was upregulated in human bone marrow derived MSC-17 when compared to MSC-[gamma]. PLAT was enriched for biological processes involving cell motility, angiogenesis, and responses to wounding. A previous study reported that IL-17A can increase MSC migration in an in vitro wound healing assay [7]. In a latter study, IL-17A was shown to enhance peripheral blood-derived MSC migration in a wound healing assay by inducing the expression of the urokinase type plasminogen activator through the activation of ERK1,2-MAPK signaling pathway [67]. Increased expression of the urokinase type plasminogen activator has been reported to facilitate MSC transendothelial migration, potentially contributing to MSC motility to sites of inflammation for tissue regeneration or immunosuppression [67]. In other studies, tissue plasminogen activators have also shown to support angiogenesis by promoting vascular endothelial cell migration to ischemic regions [68, 69]. These data suggest that MSC-17 in addition to their potent immunosuppressive properties may benefit disease conditions of ischemia injury that require tissue repair and angiogenesis.

In this study, donor to donor variation may have limited the robustness of our microarray data to detect subtle changes in MSC-17 gene expression profile. However, real-time PCR data validated changes detected in the highly regulated genes in MSC-17. More MSC-17 biological replicates may provide further insights into other genes that are differently regulated.

5. Conclusions

Enhanced expression of MHC in allogeneic MSC-[gamma] increases their immunogenicity and this may negatively impact MSC-[gamma] potency in vivo. Nevertheless, we have highlighted novel candidate immunosuppressive molecules and pathways in which MSC-[gamma] can be targeted in future studies to increase the immunomodulatory capacity of MSC. We have also identified a few novel candidate molecules that may contribute to the potent MSC-17 regulation of immune responses. These candidate molecules can be explored for their regulatory roles in MSC-17 suppression of T cell responses and in the generation of Tregs in future studies. 10.1155/2017/1025820


An earlier version of this work was presented as an abstract at the Transplantation Science Symposium, 2015, and at the American Transplant Congress, 2016.

Competing Interests

The authors declare no competing financial interests.


The authors thank the Adelaide Microarray Centre, University of Adelaide, for running the microarray samples. They also thank Dr. Philip Gregory, SA Pathology, South Australia, for proof-reading and providing feedback on this manuscript. They also thank Svjetlana Kireta and Julie Johnston for proof-reading this manuscript. This work was supported by grants from The Hospital Research Foundation, The Queen Elizabeth Hospital, Adelaide, South Australia. Kisha Nandini Sivanathan, Ph.D., was financed by the Adelaide Graduate Research Scholarship (University of Adelaide, South Australia) and The Hospital Research Foundation Scholarship (The Queen Elizabeth Hospital, Adelaide, South Australia). Kisha Nandini Sivanathan also received a young investigator grant from the 2015 Transplantation Science Symposium for this work.


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Kisha Nandini Sivanathan, (1, 2) Darling Rojas-Canales, (1, 2) Shane T. Grey, (3) Stan Gronthos, (4, 5) and Patrick T. Coates (1, 2, 6)

(1) School of Medicine, Faculty of Health Sciences, University of Adelaide, Adelaide, SA, Australia

(2) Centre for Clinical and Experimental Transplantation, Royal Adelaide Hospital, Adelaide, SA, Australia

(3) Transplantation Immunology Group, Garvan Institute of Medical Research, Sydney, NSW, Australia

(4) South Australian Health and Medical Research Institute, Adelaide, SA, Australia

(5) Mesenchymal Stem Cell Laboratory, School of Medicine, Faculty of Health Sciences, University of Adelaide, Adelaide, SA, Australia

(6) Central Northern Adelaide Renal Transplantation Service, Royal Adelaide Hospital, Adelaide, SA, Australia

Correspondence should be addressed to Kisha Nandini Sivanathan;

Received 21 September 2016; Accepted 20 December 2016; Published 15 February 2017

Academic Editor: Thomas Ichim

Caption: FIGURE 1: Principal Component Analysis (PCA) of UT-MSC, MSC-[gamma], and MSC-17. This 3-dimensional PCA graph identifies a new set of variables (PCA1, PCA2, and PCA3) that account for majority of the variability among the samples. PCA1 captures as much variability in the data as possible, PCA2 captures as much variability of the remaining variability not accounted by PCA1, and PCA3 captures as much of the remaining variability not accounted by PCA2. The symbols indicate IL-17A treated MSC, 17_; IFN-[gamma] treated MSC (Y_); and untreated-MSC (wt_). The 3 different MSC donors are indicated by C, M, and F.

Caption: FIGURE 2: Volcano plots to identify changes in gene expression between (a) MSC-17 versus UT-MSC, (b) MSC-[gamma] versus UT-MSC, and (c) MSC-17 versus MSC-[gamma]. Axes of these plots represent significance (-10 log10 p value of the ANOVA p values; y-axes) versus fold changes (linear fold change from condition pairing; x-axes). Red colour indicates upregulated genes and the green represents downregulated genes. The grey region indicates genes that were not differentially expressed and not statistically significant.

Caption: FIGURE 3: Gene expression profile of MSC-17 (1), UT-MSC (2), and MSC-[gamma] (3) from 3 MSC donors determined with Affymetrix Human Gene ST 2.0 microarrays. Supervised hierarchical clustering of genes differentially expressed between (a) MSC-[gamma] versus UT-MSC, (b) MSC17 versus UT-MSC, and (c) MSC-17 versus MSC-[gamma] determined by ANOVA p value (condition pair) p < 0.05 and fold change (linear) < -2 or >2. (a) 1278 and (b) 67 genes were differentially regulated between the treatment groups. The normalized expression value for each gene is visualised by a colour gradient: blue represents low gene expression; red represents high gene expression.

Caption: FIGURE 4: Microarray gene expression validation by RT-PCR. Gene expression of MMP1, MMP13, CCL2, CCL8, CXCL6, C3, LBP, and CH25 in MSC detected by microarray was validated by RT-PCR following 5 days of IL-17A or IFN-[gamma] treatment of human MSC. * p < 0.05 versus UT-MSC was determined by one-way ANOVA with post-Sidak multiple comparison test. Data are representative of 3 human MSC donors. Error bars depict mean [+ or -] SD.
TABLE 1: Top 30 differentially expressed genes: MSC-[gamma]
versus UT-MSC.

Gene symbol           Gene name                        mRNA Accession

Upregulated genes
  HLA-DRA             Major histocompatibility            NM_019111
                      complex, class II, DR alpha

  GBP4                Guanylate binding protein 4         NM_052941

  IDO1                Indoleamine 2,3-dioxygenase 1       NM_002164

  HLA-DRB             Major histocompatibility         ENST00000307137
                      complex, class II, DR beta

  GBP5                Guanylate binding protein 5         NM_052942

  CXCL9               Chemokine (C-X-C motif) ligand      NM_002416

  GBP2                Guanylate binding protein 2,     ENST00000464839

  SECTM1              Secreted and transmembrane 1;       NM_003004

  HLA-DRB3            Major histocompatibility         ENST00000426847
                      complex, class II, DR beta 3

  CIITA               Class II, major                     NM_00024
                      histocompatibility complex,

  GBP1                Guanylate binding protein 1,        NM_002053

  RP11-44K6.2         NULL                             ENST00000520185

  GCH1                GTP cyclohydrolase 1                NM_000161

  USP30-AS1           USP30 antisense RNA 1            ENST00000478808

  GBP2                Guanylate binding protein 2,        NM_004120
                      interferon-inducible; NULL

  HLA-DOA             Major histocompatibility            NM_002119
                      complex, class II, DO alpha;

  IFIT3               Interferon-induced protein        NM_001031683
                      with tetratricopeptide repeats

  FAM129A             Family with sequence                NM_052966
                      similarity 129, member A

  CTSS                Cathepsin S                         NM_004079

  SLC7A11             Solute carrier family 7             NM-014331
                      (anionic amino acid
                      transporter light chain, xc-
                      system), member 11

  IRF1                Interferon regulatory factor 1      NM_002198

  CD74                CD74 molecule, major              NM_001025159
                      histocompatibility complex,
                      class II invariant chain; NULL

  ICAM1               Intercellular adhesion              NM_000201
                      molecule 1

  HCP5                HLA complex P5 (nonprotein       ENST00000457127
                      coding); NULL

  LGALS17A            Charcot-Leyden crystal protein   ENST00000412609

  PARP14              Poly (ADP-ribose) polymerase       NM_017554;
                      family, member 14

  RARRES3             Retinoic acid receptor              NM_004585
                      responder (tazarotene induced)

  WARS                Tryptophanyl-tRNA synthetase;       NM_004184

  IFIT2               Interferon-induced protein          NM_001547
                      with tetratricopeptide repeats

  TMEM140             transmembrane protein 140           NM_018295

Downregulated genes
  LRRC15              Leucine rich repeat containing    NM_001135057

  KIAA1199            KIAA1199; NULL                      NM_018689

  RNU5A-8P            RNA, U5A small nuclear 8,        ENST00000364102

  COL10A1             Collagen, type X, alpha 1          NM_000493;

  COL3A1              Collagen, type III, alpha 1;        NM_000090
                      microRNA 3606

  HIST1H2A            Histone cluster 1, H2ai;            NM_003509
                      histone cluster 1, H2ah;
                      histone cluster 1, H2ag;
                      histone cluster 1, H2am;
                      histone cluster 1, H2al;
                      histone cluster 1, H2ak;
                      histone cluster 1, H3f

  SCD                 Stearoyl-CoA desaturase             NM_005063

  U2                  U2 spliceosomal RNA              ENST00000410792

  HIST1H3             Histone cluster 1, H3b;             NM_003537
                      histone cluster 1, H3f;
                      histone cluster 1, H3h;
                      histone cluster 1, H3j;
                      histone cluster 1, H3g;
                      histone cluster 1, H3i;
                      histone cluster 1, H3e;
                      histone cluster 1, H3c;
                      histone cluster 1, H3d;
                      histone cluster 1, H3a

  HIST1H1B            Histone cluster 1, H1b              NM_00532

  --                  --                               ENST00000408768

  KDELR3              KDEL (Lys-Asp-Glu-Leu)              NM_016657
                      endoplasmic reticulum protein
                      retention receptor 3

  --                  --                                  BC091525

  SNORD114-11         Small nucleolar RNA, C/D box        NR_003204

  WISP1               WNT1 inducible signaling            NM_003882
                      pathway protein 1

  U3                  Small nucleolar RNA U3           ENST00000390893

  HIST1H2BM           Histone cluster 1, H2bm             NM_003521

  COL1A1              Collagen, type I, alpha 1;          NM_000088
                      NULL histone cluster 1, H3g;
                      histone cluster 1, H3f;
                      histone cluster 1, H3b;
                      histone cluster 1, H3h;
                      histone cluster 1,

  HIST1H3             H3j; histone cluster 1, H3i;        NM_003534
                      histone cluster 1, H3e;
                      histone cluster 1, H3c;
                      histone cluster 1, H3d;
                      histone cluster 1, H3a

                      Histone cluster 1, H3f;
                      histone cluster 1, H3b;
                      histone cluster 1, H3h;
                      histone cluster 1, H3j;
                      histone cluster 1,

  HIST1H3             H3g; histone cluster 1, H3i;        NM_021018
                      histone cluster 1, H3e;
                      histone cluster 1, H3c;
                      histone cluster 1, H3d;
                      histone cluster 1, H3a

  AL732479.1          --                               ENST00000459197

  ADAM12              ADAM metallopeptidase domain       NM_003474;
                      12; NULL

  ENPP1               Ectonucleotide                      NM_006208
                      phosphodiesterase 1

  NDNF                Neuron-derived neurotrophic         NM_024574

  DHCR7               7-Dehydrocholesterol                NM_001360
                      reductase; NULL

  DHCR24              24-Dehydrocholesterol               NM_014762

  RGS4                Regulator of G-protein            NM_001102445
                      signaling 4; NULL

  CRABP2              Cellular retinoic acid binding      NM_001878
                      protein 2

  KIF20A              Kinesin family member 20A;          NM_005733

  U1                  U1 spliceosomal RNA                    --

Gene symbol           Fold change   p value

Upregulated genes
  HLA-DRA               387.78      0.00049
  GBP4                  199.41      0.00002
  IDO1                   96.72      0.00003
  HLA-DRB                89.67      0.00435
  GBP5                   88.07      0.00003
  CXCL9                  83.60      0.00002
  GBP2                   77.00      0.00004
  SECTM1                 57.59      0.00002
  HLA-DRB3               51.65      0.00868
  CIITA                  38.84      0.00003
  GBP1                   29.09      0.00001
  RP11-44K6.2            26.39      0.00060
  GCH1                   24.93      0.00022
  USP30-AS1              24.75      0.00009
  GBP2                   23.60      0.00001
  HLA-DOA                22.90      0.00003
  IFIT3                  21.49      0.00013
  FAM129A                20.89      0.00003
  CTSS                   20.10      0.00002
  SLC7A11                19.70      0.00009
  IRF1                   19.55      0.00002
  CD74                   18.49      0.00060
  ICAM1                  18.43      0.00003
  HCP5                   18.15      0.00032
  LGALS17A               18.12      0.00038
  PARP14                 17.05      0.00014
  RARRES3                17.00      0.00007
  WARS                   16.49      0.00002
  IFIT2                  16.43      0.00051
  TMEM140                16.08      0.00012

Downregulated genes
  LRRC15                -19.91      0.0007
  KIAA1199              -13.26      0.0025
  RNU5A-8P              -12.36      0.0061
  COL10A1               -12.25      0.0031
  COL3A1                -11.88      0.0000
  HIST1H2A              -11.67      0.0012
  SCD                    -9.75      0.0000
  U2                     -9.41      0.0476
  HIST1H3                -9.06      0.0053
  HIST1H1B               -8.53      0.0002
  --                     -8.25      0.0001
  KDELR3                 -8.22      0.0007
  --                     -7.87      0.0007
  SNORD114-11            -7.33      0.0021
  WISP1                  -7.18      0.0000
  U3                     -7.14      0.0128
  HIST1H2BM              -6.92      0.0024
  COL1A1                 -6.84      0.0000
  HIST1H3                -6.68      0.0032
  HIST1H3                -6.41      0.0058
  AL732479.1             -6.38      0.0015
  ADAM12                 -6.13      0.0001
  ENPP1                  -6.06      0.0002
  NDNF                   -6.00      0.0100
  DHCR7                  -5.88      0.0005
  DHCR24                 -5.84      0.0001
  RGS4                   -5.78      0.0116
  CRABP2                 -5.76      0.0015
  KIF20A                 -5.60      0.0072
  U1                     -5.38      0.0069

TABLE 2: Differentially expressed genes (mapped by DAVID): MSC-17
versus UT-MSC.

Gene symbol           Gene name                        mRNA Accession

Upregulated genes
  MMP13               Matrix metallopeptidase 13          NM_002427
                      (collagenase 3)

  C3                  Complement component 3; NULL        NM_000064

  LBP                 Lipopolysaccharide binding          NM_004139

  VMO1                Vitelline membrane outer layer      NM_182566
                      1 homolog (chicken)

  CH25H               Cholesterol 25-hydroxylase          NM_003956

  IL6                 Interleukin 6 (interferon,          NM_000600
                      beta 2); NULL

  ZC3H12A             Zinc finger CCCH-type               NM_025079
                      containing 12A

  CCL2                Chemokine (C-C motif) ligand 2      NM_002982

  ZNF253              Zinc finger protein 253             NM_021047

  SAA1                Serum amyloid A1                    NM_000331

  CXCL6               Chemokine (C-X-C motif) ligand      NM_002993

  MMP1                Matrix metallopeptidase 1           NM_002421
                      (interstitial collagenase)

  NFKBIZ              Nuclear factor of kappa light       NM_031419
                      polypeptide gene enhancer in
                      B-cells inhibitor, zeta; NULL

MIRLET7A2             MicroRNA let-7a-2                   NR_029477

  RBMY2EP             RNA binding motif protein, Y-    ENST00000444169
                      linked, family 2, member E

  CCL8                Chemokine (C-C motif) ligand 8      NM_005623

  STC1                Stanniocalcin 1                     NM_003155

  SFRP4               Secreted frizzled-related           NM_003014
                      protein 4

  SLC22A3             Solute carrier family 22            NM_021977
                      (extraneuronal monoamine
                      transporter), member 3

  TTTY11              Testis-specific transcript,         NR_001548
                      Y-linked 11 (nonprotein

  STEAP2              STEAP family member 2,            NM_001244944
                      metalloreductase; NULL

  SCARNA18            Small Cajal body-specific RNA       NR_003139

LOC100287834          Uncharacterised LOC100287834        NR_028349

Downregulated genes

  RPS24               Ribosomal protein S24; NULL       NM_001142285

LOC100133299          GALI1870                            AY358688

  POU5F1              POU class 5 homeobox 1           ENST00000259915

  TMEM171             Transmembrane protein 171           NM_173490

  IGLJ2               Immunoglobulin lambda joining    ENST00000390322

  ITGA6               Integrin, alpha 6; NULL          ENST00000264107

  RNU7-25P            RNA, U7 small nuclear 25         ENST00000516544
                      pseudogene; RNA, U7 small
                      nuclear 11 pseudogene

  GTF2IRD2B           GTF2I repeat domain containing    NM_001003795

  SERTAD4             SERTA domain containing 4        ENST00000367012

  TPTE                Transmembrane phosphatase with   ENST00000415664
                      tensin homology

Gene symbol           Fold change   p value

Upregulated genes
  MMP13                  15.60      0.0021
  C3                     11.56      0.0039
  LBP                    5.35       0.0031
  VMO1                   4.07       0.0022
  CH25H                  3.99       0.0023
  IL6                    3.44       0.0083
  ZC3H12A                3.09       0.0010
  CCL2                   3.08       0.0405
  ZNF253                 2.82       0.0010
  SAA1                   2.72       0.0102
  CXCL6                  2.44       0.0014
  MMP1                   2.40       0.0356
  NFKBIZ                 2.36       0.0232
MIRLET7A2                2.30       0.0031
  RBMY2EP                2.27       0.0278
  CCL8                   2.20       0.0012
  STC1                   2.20       0.0023
  SFRP4                  2.19       0.0136
  SLC22A3                2.15       0.0452
  TTTY11                 2.15       0.0252
  STEAP2                 2.12       0.0225
  SCARNA18               2.06       0.0254
LOC100287834             2.06       0.0328

Downregulated genes
  RPS24                  -2.01      0.0209
LOC100133299             -2.03      0.0095
  POU5F1                 -2.04      0.0129
  TMEM171                -2.05      0.0133
  IGLJ2                  -2.07      0.0252
  ITGA6                  -2.11      0.0109
  RNU7-25P               -2.16      0.0047
  GTF2IRD2B              -2.20      0.0040
  SERTAD4                -2.29      0.0470
  TPTE                   -2.85      0.0128

TABLE 3: Top 30 differentially expressed genes: MSC-17 versus

Gene symbol       Gene name                          mRNA Accession

  MMP13           Matrix metallopeptidase 13           NM-002427
                  (collagenase 3)

  HIST1H2AI       Histone cluster 1, H2ai              NM_003509

  U3              Small nucleolar RNA U3            ENST00000390893

  ZNF25           Zinc finger protein 25            ENSG00000175395

  LRRC15          Leucine rich repeat containing      NM_001135057

  HIST1H3G        Histone cluster 1, H3g               NM_003534

  SNORD114-11     Small nucleolar RNA, C-D box         NR_003204

  HIST1H3B        Histone cluster 1, H3b               NM_003537

  U1              U1 spliceosomal RNA                NONHSAT054977

  HIST1H1B        Histone cluster 1, H1b               NM_005322

  SCD             Stearoyl-CoA desaturase              NM_005063

  KRT16P4         Keratin 16 pseudogene 4           ENST00000453883

  ADAM12          ADAM metallopeptidase domain        NM_001288973

  HIST1H2BM       Histone cluster 1, H2bm              NM_003521

  HIST1H3F        Histone cluster 1, H3f               NM_021018

  ADAM12          ADAM metallopeptidase domain        NM_001288973

  KIAA1199        KIAA1199; NULL                       NM_018689

  COL10A1         Collagen, type X, alpha 1            NM_000493

  DHCR7           7-Dehydrocholesterol                 NM_001360
                  reductase; NULL

  P4HA3           Prolyl 4-hydroxylase, alpha          NM_182904
                  polypeptide III

  LBP             Lipopolysaccharide binding           NM_004139

  HAS1            Hyaluronan synthase 1                NM_001523

  COL1A1          Collagen, type I, alpha 1;           NM_000088

  NDNF            Neuron-derived neurotrophic          NM_024574

  ELN             Elastin; NULL                        NM_000501

  WISP1           WNT1 inducible signaling             NM_003882
                  pathway protein 1

  ADAM12          ADAM metallopeptidase domain         NM_003474
                  12; NULL

  KDELR3          KDEL (Lys-Asp-Glu-Leu)               NM_016657
                  endoplasmic reticulum protein
                  retention receptor 3

  HIST1H3I        Histone cluster 1, H3i               NM_003533

  CNN1            Calponin 1, basic, smooth            NM_001299

  HLA-DRA         Major histocompatibility              NM_019111
                  complex, class II, DR alpha

  GBP4            Guanylate binding protein 4          NM_052941

  HLA-DRA         Major histocompatibility          ENST00000442960
                  complex, class II, DR alpha;

  CXCL9           Chemokine (C-X-C motif) ligand       NM_002416

  IDO1            Indoleamine 2,3-dioxygenase 1        NM_002164

  HLA-DRB3        Major histocompatibility          ENST00000307137
                  complex, class II, DR beta 3

  GBP5            Guanylate binding protein 5           NM_052942

  SECTM1          Secreted and transmembrane 1;        NM_003004

  GBP2            Guanylate binding protein 2,      ENST00000464839

  HLA-DPA1        Major histocompatibility            NM_001242524
                  complex, class II, DP alpha 1

  IFIT3           Interferon-induced protein          NM_001031683
                  with tetratricopeptide repeats

  CIITA           Class II, major                      NM_000246
                  histocompatibility complex,

  GBP1            Guanylate binding protein 1,         NM_002053

  PSAT1           Phosphoserine aminotransferase       NM_021154

  HLA-DPB1        NULL                             OTTHUMT00000310634

  GBP1P1          Guanylate binding protein 1,      ENST00000513638
                  pseudogene 1

  GCH1            GTP cyclohydrolase 1                 NM_000161

  PARP14          Poly (ADP-ribose) polymerase         NM_017554
                  family, member 14

  IRF1            Interferon regulatory factor 1       NM_002198

  HLA-DOA         Major histocompatibility             NM_002119
                  complex, class II, DO alpha

  RARRES3         Retinoic acid receptor               NM_004585
                  responder (tazarotene induced)

  LGALS17A        Charcot-Leyden crystal protein    ENST00000412609

  HLA-DOA         Major histocompatibility             NM_002119
                  complex, class II, DO alpha

  SLC7A11         Solute carrier family 7              NM-014331
                  (anionic amino acid
                  transporter light chain, xc-
                  system), member 11

  USP30-AS1       USP30 antisense RNA 1             ENST00000478808

  HCP5            HLA complex P5 (non-protein       ENST00000457127
                  coding); NULL

  ICAM1           Intercellular adhesion               NM_000201
                  molecule 1

  WARS            Tryptophanyl-tRNA synthetase;        NM_004184

  APOL1           Apolipoprotein L, 1; NULL            NM_003661

  ERVK-7          Endogenous retrovirus group K,    ENST00000522373
                  member 7; novel transcript

  HLA-DPB2        Major histocompatibility             NR_001435
                  complex, class II, DP beta 2

Gene symbol       Fold change   p value

  MMP13              24.27       0.0009
  HIST1H2AI          17.44       0.0005
  U3                 13.96       0.0118
  ZNF25              13.66       0.0008
  LRRC15             13.57       0.0005
  HIST1H3G           12.68       0.0008
  SNORD114-11        12.33       0.0386
  HIST1H3B           11.20       0.0064
  U1                 10.54       0.0017
  HIST1H1B           10.28       0.0003
  SCD                10.15       0.0008
  KRT16P4            9.11        0.0122
  ADAM12             8.96        0.0031
  HIST1H2BM          8.87        0.0125
  HIST1H3F           8.77        0.0081
  ADAM12             8.70        0.0121
  KIAA1199           8.48        0.0045
  COL10A1            6.87        0.0136
  DHCR7              6.51        0.0022
  P4HA3              6.49        0.0043
  LBP                6.11        0.0021
  HAS1               6.10        0.0038
  COL1A1             5.94       0.000002
  NDNF               5.75        0.0147
  ELN                5.65        0.0014
  WISP1              5.61        0.0052
  ADAM12             5.56        0.0008
  KDELR3             5.36        0.0085
  HIST1H3I           5.02        0.0014
  CNN1               4.69       0.00001

  HLA-DRA           -553.64      0.0005
  GBP4              -244.27      0.0001
  HLA-DRA           -188.10     0.00005
  CXCL9             -96.44      0.00001
  IDO1              -76.98      0.00004
  HLA-DRB3          -70.87       0.0024
  GBP5              -65.02      0.000004
  SECTM1            -50.74       0.0001
  GBP2              -41.55      0.00001
  HLA-DPA1          -40.79       0.0011
  IFIT3             -39.52       0.0011
  CIITA             -37.92      0.000003
  GBP1              -35.79       0.0002
  PSAT1             -31.20       0.0001
  HLA-DPB1          -28.81      0.00002
  GBP1P1            -27.87      0.00001
  GCH1              -26.74       0.0002
  PARP14            -26.49       0.0008
  IRF1              -24.92      0.000003
  HLA-DOA           -24.00      0.00001
  RARRES3           -23.10       0.0001
  LGALS17A          -22.45       0.0003
  HLA-DOA           -21.85      0.00001
  SLC7A11           -21.62      0.00004
  USP30-AS1         -20.00       0.0001
  HCP5              -19.90       0.0001
  ICAM1             -19.63       0.0001
  WARS              -19.41       0.0001
  APOL1             -19.36      0.00002
  ERVK-7            -18.93       0.0267
  HLA-DPB2          -18.91      0.00009
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Article Details
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Title Annotation:Research Article; Interleukin-17A
Author:Sivanathan, Kisha Nandini; Rojas-Canales, Darling; Grey, Shane T.; Gronthos, Stan; Coates, Patrick T
Publication:Stem Cells International
Article Type:Report
Date:Jan 1, 2017
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