Integrating the lncRNA and mRNA expression proles of TLR4-primed mesenchymal stem cells in ankylosing spondylitis

Our previous study found that the toll-like receptor 4 (TLR4) expression of ankylosing spondylitis (AS) patients was signicantly different from that of healthy donors. The goals of this study were to explore the expression proles and functional networks of lncRNAs and mRNAs in TLR4-primed mesenchymal stromal cells from AS patients (AS-MSCs) and to clarify the mechanisms by which TLR4-primed MSCs exert immunoregulatory effects in AS. of density gradient centrifugation method and then labelled with carboxyuorescein diacetate succinimidyl ester (CFSE; BD Bioscience, USA). PBMCs were incubated with 5 μM CFSE in phosphate-buffered saline (PBS) at 37°C for 5 mins. After the incubation, the staining reaction was terminated by washing the PBMCs with complete medium twice. For co-culture with AS-MSCs (effector cells), AS-MSCs at a density of 1×10 5 cells/well seeded in 6-well plates were stimulated with or without 1 µg/ml LPS for 4 hours and then subjected to Co-60 irradiation (30 Gy). CFSE-labelled PBMCs (responder cells) at a cell density of 1×10 6 were then added to the MSC cultures. The co-cultures were stimulated with human anti-CD3/CD28 monoclonal antibodies (mAbs; CD3: 200 ng/ml; CD28: 1 µg/ml, BD Bioscience, USA) for 5 days, after which the PBMCs were harvested and stained with an anti-CD4-FITC antibody (BD Bioscience, USA), and cell proliferation was evaluated using ow cytometry. A minimum of 10,000 live cell events gated in scatter plots were analysed for each sample. analyses examine roles of lncRNAs TLR4-primed


Abstract Background
Our previous study found that the toll-like receptor 4 (TLR4) expression of ankylosing spondylitis (AS) patients was signi cantly different from that of healthy donors. The goals of this study were to explore the expression pro les and functional networks of lncRNAs and mRNAs in TLR4-primed mesenchymal stromal cells from AS patients (AS-MSCs) and to clarify the mechanisms by which TLR4-primed MSCs exert immunoregulatory effects in AS.

Methods
Firstly, the immunoregulatory effects of MSCs were determined after TLR4 activation. Then, the differentially expressed (DE) lncRNAs and mRNAs between the control group (AS-MSCs without stimulation) and experimental group (AS-MSCs stimulated with lipopolysaccharide) were identi ed through high-throughput sequencing followed by qRT-PCR con rmation. Finally, bioinformatic analyses were performed to identify the critical biological functions, signalling pathways and associated functional networks involved in the TLR4-primed immunoregulatory function of AS-MSCs.

Results
TLR4-primed AS-MSCs showed a strong ability to inhibit the proliferation of peripheral blood mononuclear cells (PBMCs) with 1 µg/ml LPS stimulation for 4 hours. A total of 147 DE lncRNAs and 698 DE mRNAs were identi ed between TLR4-primed AS-MSCs and unstimulated AS-MSCs.
Signi cant fold changes in lncRNA and mRNA levels were con rmed by qRT-PCR. GO and KEGG analysis demonstrated that the DE mRNAs and lncRNAs were highly associated with the in ammatory response. Cis-regulation prediction revealed 9 novel lncRNAs while trans-regulation prediction revealed 15 lncRNAs, respectively.

Conclusions
Our research describes the lncRNA and mRNA expression pro les and functional networks in TLR4-primed AS-MSCs, which is supposed to enhance the understanding of the pathogenesis of AS-MSC immunoregulatory dysfunction.

Background
Ankylosing spondylitis (AS) is a chronic in ammatory rheumatic disease characterized by in ammatory back pain and asymmetrical peripheral arthritis [1]. Previous studies have shown that AS is closely related to immune dysfunction. However, the pathogenesis of AS is largely unknown. Mesenchymal stem cells (MSCs) are a group of self-renewing cells that have a signi cant immunomodulatory ability that allows them to regulate T cell proliferation and differentiation and inhibit dendritic cell (DC) maturation [2,3]. According to recent studies, abnormal immunoregulation by MSCs can lead to several autoimmune diseases, such as immune thrombocytopenia and systemic lupus erythematosus (SLE) [4][5][6]. In our previous study, we found impairment in the immunoregulatory functions of MSCs from AS patients, which might play an important role in the pathogenesis of AS [7]. Furthermore, our clinical trial demonstrated that intravenous infusion of MSCs was a feasible, safe, and effective approach for the treatment of AS [8]. However, our understanding of the immunoregulatory function of MSCs is still in its infancy, and further characterization and identi cation of key factors regulating these properties are still needed.
Toll-like receptors (TLRs) have been demonstrated to play important roles in regulating the immunomodulatory properties of MSCs, but inconsistent results have been reported [9]. Liotta et al. found that ligation of TLR4 suppressed the inhibitory effects of human bone marrow (BM)-MSCs on T cell proliferation by downregulating Jagged-1 expression [10]. Waterman et al. demonstrated that MSCs could be primed towards a proin ammatory phenotype after TLR4 activation [11]. In contrast, Opitz et al. reported that TLR4 enhanced the immunosuppressive properties of human BM-MSCs by directly inducing indoleamine 2,3-dioxygenase 1 (IDO1) [12]. Our previous study found that the expression of TLR4 in MSCs was downregulated in AS patients compared to healthy donors (HDs). The inhibitory effects of MSCs on CD4 + T cell proliferation in AS were enhanced by activation of TLR4. It has been suggested that TLR4 plays a signi cant role in regulating the immunomodulatory ability of MSCs in AS (AS-MSCs) [13]. However, the speci c immunoregulatory mechanisms by which TLR4 controls MSC immunoregulation must be addressed.
AS-MSCs and could provide potential targets to improve the curative effect of MSCs on AS.

AS-MSC isolation and cell culture
MSCs were isolated from BM aspirates taken from AS patients who provided informed consent through density gradient centrifugation, as described in our previous study [18]. After density gradient centrifugation, MSCs were isolated through plastic adherence and grown at 37°C in an atmosphere of 5% CO2 for one week. The MSCs were trypsinized when the cultures reached 80-90% con uence. MSCs in passages 3-5 were used in subsequent experiments. MSCs were identi ed on the basis of immunological phenotypes and the triple-lineage differentiation capability, as previously described [18]. After identifying MSC immunophenotypic markers by ow cytometry, cells in passages three to ve were used for subsequent experiments.

Pre-stimulation of TLR4 on AS-MSCs
To stimulate TLR4 on MSCs effectively, determining the proper stimulating concentration and treatment time of the TLR4 ligand (lipopolysaccharide, LPS) was necessary. For the time-based stimulation test, 1.0 µg/ml LPS (Sigma-Aldrich, USA) was added to the culture medium for different times (0, 2, 4, 8, 12 and 24 hours) before a mixed lymphocyte reaction (MLR) was performed. For the concentration-based stimulation test, different concentrations (0, 0.1, 1 and 10 µg/ml) of LPS were added to cultured cells for the previously selected time. The best conditions for MSC TLR4 activation were evaluated by assessing the level of p38 phosphorylation by western blotting. After determining the best stimulation time and concentration of LPS, MSCs were pre-primed before co-culture with peripheral blood mononuclear cells (PBMCs) and gene analysis.
Co-culture of AS-MSCs and PBMCs for PBMC proliferation analysis AS-MSCs were primed with 1 µg/ml LPS for 4 hours prior to co-culture with PBMCs to activate TLR4. PBMCs were harvested from blood samples taken from healthy donors using the Ficoll-Paque density gradient centrifugation method and then labelled with carboxy uorescein diacetate succinimidyl ester (CFSE; BD Bioscience, USA). PBMCs were incubated with 5 μM CFSE in phosphate-buffered saline (PBS) at 37°C for 5 mins. After the incubation, the staining reaction was terminated by washing the PBMCs with complete medium twice. For co-culture with AS-MSCs (effector cells), AS-MSCs at a density of 1×10 5 cells/well seeded in 6-well plates were stimulated with or without 1 µg/ml LPS for 4 hours and then subjected to Co-60 irradiation (30 Gy). CFSE-labelled PBMCs (responder cells) at a cell density of 1×10 6 were then added to the MSC cultures. The co-cultures were stimulated with human anti-CD3/CD28 monoclonal antibodies (mAbs; CD3: 200 ng/ml; CD28: 1 µg/ml, BD Bioscience, USA) for 5 days, after which the PBMCs were harvested and stained with an anti-CD4-FITC antibody (BD Bioscience, USA), and cell proliferation was evaluated using ow cytometry. A minimum of 10,000 live cell events gated in scatter plots were analysed for each sample.
Library construction and high-throughput sequencing Three MSC samples from AS patients were separated into two subgroups: MSCs cultured without stimulation (control group, samples C1-C3) and MSCs stimulated with 1 μg/ml LPS for four hours to activate TLR4 speci cally (experimental group, samples T1-T3). The total RNA concentration was quanti ed with a NanoDrop ND-2000 (Thermo Scienti c), and RNA integrity was assessed using an Agilent Bioanalyzer 2100 (Agilent Technologies). Sample labelling, microarray hybridization and washing were performed based on the manufacturer's standard protocols. Brie y, total RNA was transcribed into double-stranded complementary DNA (cDNA), which was then synthesized into complementary RNA (cRNA) and labelled with Cyanine-3-CTP. The labelled cRNAs were hybridized onto the microarray. After washing, the arrays were scanned by an Agilent Scanner G2505C (Agilent Technologies). The Agilent Human lncRNA Microarray 2018 (4*180k, Design ID: 085630) was used in this experiment, and data analysis of the 6 samples was conducted by OE Biotechnology Co., Ltd. (Shanghai, China).

Expression analysis
Feature Extraction software (version 10.7.1.1, Agilent Technologies) was used to analyse array images to obtain raw data. GeneSpring (version 14.8, Agilent Technologies) was employed to complete the basic analysis with the raw data. First, the raw data were normalized with the quantile algorithm. The probes with at least 1 condition out of 2 conditions having ags in "Detected" were chosen for further data analysis. DE genes were then identi ed through fold change data as well as the P value calculated with a t-test. The threshold set for up-and downregulated genes was a fold change ≥ 2.0 and a P value ≤ 0.05. Afterwards, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were applied to determine the roles of these differentially expressed (DE) mRNAs. Finally, hierarchical clustering was performed to display the distinguishable gene expression patterns among samples.

qRT-PCR validation
To validate the reliability of high-throughput RNA-seq and explore the expression trends of mRNAs and lncRNAs, we performed quantitative real-time PCR (qRT-PCR) for biological validation. Total RNA was isolated from AS-MSCs with or without LPS stimulation using TRIzol according to the manufacturer's protocol. cDNA was transcribed using a PrimeScript RT reagent kit (Takara, Otsu). qRT-PCR was then performed, and the data were analysed using the 2 -DDCt method. The primer sequences used in the qRT-PCR assay are provided in Supplementary Table S1.

Cis-and trans-regulation predictions of DE lncRNAs
It has been suggested that lncRNAs regulate gene expression through both cis-and trans-regulation. For cis-regulation prediction, we identi ed each paired lncRNA and mRNA by the following procedures: (1) the mRNA loci were within 100-kb windows upstream or downstream of the given lncRNA, and (2) the Pearson correlation of lncRNA-mRNA expression was signi cant (P ≤ 0.05). For trans-regulation prediction, we enriched the coexpressed mRNAs with DE lncRNAs that signi cantly overlapped with the host genes of transcription factors (TFs). Using the threshold P < 0.05, each lncRNA could be connected with one to more than a dozen TFs, and each pair of lncRNA-TF was the result of several gene enrichments based on the hypergeometric cumulative distribution function. Then, we constructed the lncRNA-TF-mRNA network using Cytoscape software.

Statistical analysis
Data are expressed as the mean ± standard deviation (SD) and were analysed using the statistical software package SPSS16.0. Pearson correlation was used in lncRNA-mRNA co-expression analyses. A p value < 0.05, fold enrichment > 2, and log 2 FC > 1 were considered statistically signi cant.

Results
The effect of TLR4 activation on AS-MSCs is time and dose dependent To investigate whether the activation of TLR4 in AS-MSCs can affect the immunoregulatory ability of these cells, AS-MSCs were pre-stimulated with LPS before being co-cultured with PBMCs. To determine the best stimulation time and concentration for the TLR4 ligand used, the level of p38 phosphorylation was examined by western blotting. MSCs were rst exposed to LPS at a concentration of 1 µg/ml for the indicated times (0, 2, 4, 8, 12 and 24 hours) and then treated with three different concentrations (0, 0.1, 1 and 10 µg/ml) of stimuli for the previously selected time. The upregulation of the phospho-p38 level was highest at 4 hours with the LPS concentration of 1 µg/ml and declined thereafter (Fig. 1A).
TLR4-primed AS-MSCs demonstrate an enhanced inhibitory effect on CD4 + T cell proliferation Previous reports have shown that co-culture of unprimed MSCs with PBMCs can inhibit PBMC proliferation and/or activation [19]. Thus, we sought to assess the potential in uence of TLR4 activation on the immunoregulatory effect of MSCs derived from AS patients. To this end, MSC-PBMC coculturing was conducted with CFSE-labelled PBMCs (responder cells) co-cultured with unprimed or TLR4-primed MSCs (effector cells) for 5 days, after which time the proliferating responders were sorted by ow cytometry for CD4 positivity and then gated on CFSE expression. As shown in Fig.  1C, 72.8% of the CD4 + T cells underwent proliferation when cultured without MSCs, but this proportion was reduced to 46.3% when cultured with AS-MSCs. Activation of TLR4 with 1 μg/ml LPS signi cantly enhanced the immunoregulatory effect of AS-MSCs, which reduced CD4 + T cell proliferation to 40.3% (p < 0.05) (Fig. 1B). qRT-PCR results suggested that the expression of several cytokines and chemokines (TNF-α, CXCL-9, PDL1, IL-1β, IL-6 and iNOS) was strengthened after stimulation with LPS (Fig. 1C).

Identi cation of DE mRNAs and lncRNAs
A total of 698 mRNAs were DE in TLR4-primed MSCs compared to unprimed MSCs from AS patients. Among these genes, 594 mRNAs were upregulated, and 104 mRNAs were downregulated. The DE mRNAs are depicted using a clustergram ( Fig. 2A) and volcano plots (Fig. 2C). The 20 mRNAs with the largest fold changes are shown in Table 1. Several immunoregulatory cytokines and chemokines, such as CXCL10, CXCL11, IDO1, CXCL8, CXCL1, CCL20, IL6 and SOD2, which are secreted by MSCs and play important roles in regulating immunocytes, were included in this list. A total of 147 lncRNAs, including 107 upregulated and 40 downregulated lncRNAs, were differentially expressed in TLR4-primed MSCs compared to unprimed MSCs from AS patients. The DE lncRNAs are depicted in a clustergram (Fig. 2B) and volcano plots (Fig. 2D). The 10 lncRNAs with the largest fold changes are shown in Table 2.
Validation of DE mRNA and lncRNA expression levels To con rm the RNA-seq results, several important DE mRNAs and lncRNAs were assessed by qRT-PCR. We found that the expression of mRNAs (CXCL1, CXCL8, CXCL10, CXCL11 and CCL20) and lncRNAs (MIR3142HG, LOC105371619, LOC105374444, PACERR and LOC105375914) was signi cantly upregulated in TLR4-primed AS-MSCs compared to unstimulated AS-MSCs (P<0.05) ( Fig. 3A and 3B). All qRT-PCR results were consistent with the RNA-seq results, con rming the reliability of the sequencing data.

GO and KEGG analyses
We performed GO analysis of the DE mRNAs and lncRNAs. The top 10 GO terms related to biological processes, cellular components and molecular functions are provided in Fig. 4A and supplementary table 1. In the biological process category, the top 5 GO terms associated with DE mRNAs were defence response to virus, type I interferon signalling pathway, interferon-gamma-mediated signalling pathway, in ammatory response and response to virus. In the molecular function category, the top 5 GO terms associated with the DE mRNAs were chemokine activity, CXCR chemokine receptor binding, ubiquitin-protein transferase activity, double-stranded RNA binding and TF activity, and sequence-speci c DNA binding. In the cellular component category, the top 5 GO terms associated with the DE mRNAs were cytoplasm, cytosol, extracellular space, nucleus and extracellular region. KEGG analysis of the DE mRNAs determined that 75 pathways were signi cantly altered in TLR4-primed MSCs from AS patients. The top 30 affected pathways are shown in Fig. 4B. The top 10 pathways and DE mRNAs associated with these pathways are shown in supplementary table 2. The top pathways included the NOD-like receptor (NLR) signalling pathway, the TNF signalling pathway, the NF-kappa B signalling pathway, cytokine-cytokine receptor interaction, the IL-17 signalling pathway and the TLR signalling pathway, which contribute to the immunoregulatory function of AS-MSCs.

Interaction and co-expression network analyses
The interactions between proteins encoded by DE mRNAs are shown in Fig. 4C. OAS2, OAS3, OAS1, OASL, STAT1, IRF9, HLA-F, IRF1, IRF2 and HERC5 were identi ed as key genes that interact with many other DE mRNAs in this network. Based on the expression levels of DE lncRNAs and DE mRNAs, the PCC describing the co-expression association between 147 DE lncRNAs and 698 DE mRNAs was calculated. A total of 1,072 DE lncRNA-DE mRNA co-expression pairs were obtained with an absolute PCC value ≥ 0.85 and P < 0.05. Among these pairs, 706 lncRNA-mRNA pairs were identi ed as being positively co-expressed, whereas 366 lncRNA-mRNA pairs were found to be negatively co-expressed (Fig. 4D).

Cis-regulation prediction of DE lncRNAs
Cis-regulation, which regulates the transcription of nearby genes located on the same chromosome, is vital for gene expression. A total of 9 lncRNA transcripts and their predicted cis-regulated protein-coding genes were identi ed in the top 20 cis-regulated genes (Fig. 5A). POU6F2-AS2 was predicted to cis-regulate POU6F2, LOC107986962 was predicted to cis-regulate GRHL2, PIK3CD-AS1 and PIK3CD-AS2 were predicted to cis-regulate SLC26A4, LOC105376617 was predicted to cis-regulate C11orf91, GRM7-AS1 was predicted to cis-regulate ADAMTS9, GRM7-AS2 was predicted to cis-regulate ADAMTS10 and ADAMTS11, GRM7-AS3 was predicted to cis-regulate ADAMTS12 and ADAMTS13, and LOC102723716 was predicted to cis-regulate FGFR1. These networks may provide valuable clues about these lncRNAs and their nearby coding genes in the development of AS.

Trans-regulation prediction of DE lncRNAs
One of the important mechanisms by which lncRNAs function is by participating in particular pathways regulated by TFs. A top 500 lncRNA-TF network, which showed that 15 lncRNAs participate in pathways regulated by TFs, was constructed to provide key data for subsequent research (Fig. 5B). Then, we selected the abovementioned relationships among the lncRNAs and TFs and further introduced the target mRNAs to build the lncRNA-TF-mRNA network (Fig. 5C).

Discussion
In our present research, we utilized high-throughput sequencing followed by bioinformatic analysis to analyse the mRNA and lncRNA expression pro les and functional networks of TLR4-primed AS-MSCs. These ndings were then con rmed by qRT-PCR. KEGG pathway analysis indicated that some key pathways, such as the NF-kappa B and TLR signalling pathways, might contribute to the immunoregulatory function of TLR4-primed AS-MSCs. In addition, we obtained novel ndings by bioinformatic analyses of DE transcripts, including identi cation of the most signi cantly altered GO categories, construction of a co-expression network for lncRNA function prediction, and cis-and trans-regulation predictions of lncRNAs. Our results provide a model that can be used to explore the roles of lncRNAs and mRNAs in the immunoregulatory mechanisms of TLR4-primed AS-MSCs.

MSCs are one of the most important immunoregulatory cell types and regulate the functions of many immune cells, including T cells, B cells and
DCs [20][21][22]. Abnormal immunoregulation by MSCs can lead to several autoimmune diseases [4]. Moreover, MSCs exert considerable therapeutic effects on several autoimmune diseases owing to their multilineage differentiation potential and highly immunoregulatory properties [23,24]. In our previous study, we found that impairment in the immunoregulatory functions of MSCs played a key role in the pathogenesis of AS [7]. Additionally, our clinical trial study demonstrated that infusion of MSCs isolated from healthy individuals is a safe and e cient method for the treatment of AS [8].
Accumulating evidence suggests that TLR activation can modulate the immunoregulatory functions of MSCs [10][11][12]. In addition, emerging evidence further suggests a role for TLRs in the pathogenesis of spondyloarthropathies, including AS [25]. According to our previous study, the expression of TLR4 was downregulated in MSCs derived from AS patients, and compared with MSCs from healthy donors, TLR4-primed AS-MSCs possessed enhanced immunoregulatory effects limiting the proliferation of naive CD4 + T cells [26]. However, the precise mechanism underlying the enhanced immunoregulatory ability of TLR4-primed AS-MSCs remains unclear. Therefore, we measured the differential expression pro les of lncRNAs and mRNAs in AS-MSCs after TLR4 activation to identify the regulatory network of lncRNAs and mRNAs in these cells. The results showed that there were 698 DE mRNAs and 147 DE lncRNAs in TLR4-primed AS-MSCs compared with unstimulated cells. The top 5 mRNAs and lncRNAs, which may be involved in the regulatory dysfunction of AS-MSCs, were veri ed by qRT-PCR. mRNA expression pro les re ect the biological behaviours and functions of cells. In this study, KEGG pathway analysis revealed that 75 signalling pathways exhibited signi cant differences between TLR4-primed AS-MSCs and unstimulated AS-MSCs. Among these pathways, the NLR signalling pathway, the TNF signalling pathway, the NF-kappa B signalling pathway, cytokine-cytokine receptor interaction and the TLR signalling pathway were prominent. Recent studies indicate that the activation of TLRs can activate NF-κB and MAPK signalling pathways to promote the secretion of pro-in ammatory cytokines, such as IL-6, IL-12, TNF-α and type I IFNs, which drive in ammation in AS [27]. Consistent with these ndings, we found that the expression of TNF-α, IL-6 and IL1β in AS-MSCs was signi cantly increased after TLR4 activation, which supported the crucial roles for TLR4 in the NF-κB and MAPK signalling pathways identi ed by microarray analysis. In addition, as pathogen recognition receptors, both TLR and NLR activate pathways mediated through different adaptor proteins that are commonly found to activate NF-κB [28]. The NF-κB-mediated activation of MSCs leads to the secretion of TNF-α and other cytokines. Elevated pro-in ammatory cytokine levels are one of the main manifestations of AS, as con rmed by previous research. Our results further con rmed the signi cant role of TLR4 in the pathology of AS. The selected top DE mRNAs and lncRNAs included in the NF-κB pathway might be possible upstream targets of pathological in ammation in AS. mRNA expression pro les are under the control of a series of epigenetic regulators, of which lncRNAs are an important component [15,29]. In recent years, an increasing number of lncRNAs have been reported to perform key roles in the pathogenesis and development of AS. For example, lncRNA-AK001085 expression was found to be downregulated in AS patients, which served as a potential diagnostic indicator; thus, this lncRNA is considered a potential suppressor of AS [30]. Our previous microarray study identi ed four lncRNAs (lnc-ZNF354A-1, lnc-LIN54-1, lnc-FRG2C-3 and lnc-USP50-2) that are involved in the abnormal osteogenic differentiation of AS-MSCs [31]. However, the immunoregulatory function of AS-MSCs regulated by lncRNAs has not been explored. Our research identi es the lncRNA expression pro le in an in ammatory environment based on previous studies, which provides a possible way to further explore the regulatory function of lncRNAs in AS. To the best of our knowledge, this study is the rst to use microarray analyses to examine the roles of lncRNAs in TLR4-primed AS-MSCs.
In this study, several lncRNAs with the largest fold changes among DE lncRNAs were studied. For example, GBP1P1 is a lncRNA that acts as a prognostic biomarker for hepatocellular carcinoma [32]. MIR3142HG can regulate the in ammatory response following IL-1β-mediated activation of human lung broblasts, which is a positive regulator of IL-8 and CCL2 release [33]. The in ammatory response regulation by MIR3142HG indicates that it may contribute to the enhanced immunoregulatory ability of TLR4-primed AS-MSCs and the immunoregulatory dysfunction seen in AS.
Unfortunately, most of the DE lncRNAs, such as LOC101926887, LOC105378410, and LOC107984251, have not been studied yet. Further exploration is needed in the future.
Regulatory lncRNAs act in a cis and/or trans manner to in uence or interact with nearby or distant genes [34]. In our study, 9 lncRNA transcripts were predicted to cis-regulate nearby protein-coding genes. In addition, we predicted the functions of trans-regulatory lncRNAs using TFs that regulate protein-coding gene expression. LncRNAs in the core lncRNA-TF-mRNA network were grouped into 8 categories of target mRNAs: SOD2, IFIT3, IFIT5, MICB, SP140L, GBP2, PTGS2 and HIVEP2. SOD2 is a component of antioxidant defence systems, which are crucial in defending cells against oxidative stress. SOD2-overexpressing BM-MSCs have an enhanced therapeutic effect on brain injury treatment in traumatic brain injury mice [35]. Following exposure to systemic sclerosis patient serum, MSCs enhance the expression of the SOD2 antioxidant gene to adapt to the oxidative environment and exert their therapeutic effect [36]. Exposure to LPS induces oxidative stress in AS-MSCs, so SOD in AS-MSCs may be crucial to defend cells against oxidative stress and to exert the immunoregulatory effects of the AS-MSCs. In addition, 15 lncRNAs were identi ed to function in a trans-regulation manner, but the underlying mechanisms of how "lncRNA-TF-target gene" networks affect TLR4-induced immunoregulation remain to be identi ed. We expect to integrate lncRNAs into the trans-regulatory network, which will help us to understand the transcriptional control of TFs. The above mentioned 24 DE lncRNA and nearby cis/trans target DE mRNA pairs identi ed in the present study provide novel information for understanding the biological functions of lncRNAs in AS-MSCs.
Our study has several limitations. First, RNA-seq is an important method to screen possible lncRNAs and mRNAs associated with speci c diseases, but the results of big-data analyses may include false positives. Therefore, we performed qRT-PCR to further verify differential expression. Second, we predicted lncRNA functions only indirectly using bioinformatic analysis and validated several DE lncRNAs. Further functional studies on the mechanism are warranted to clarify the roles of lncRNAs.      Figure 1 The immunoregulatory function of TLR4-primed AS-MSCs. A: To determine the best duration and concentration of TLR4 ligand pre-stimulation, we examined the level of p38 phosphorylation by western blotting. MSCs were exposed to LPS at four different concentrations (0 µg/ml, 0.1 µg/ml, 1 µg/ml, and 10 µg/ml) for 4 hours or treated at the concentration of 1 µg/ml for the indicated time (0 hours, 2 hours, 4 hours, 8 hours, 12 hours or 24 hours). The upregulation of the phospho-p38 level was highest at 4 hours with the concentration of 1 µg/ml LPS and declined thereafter. B: AS-MSCs were pre-stimulated with or without 1 µg/ml LPS for 4 hours and then co-cultured with PBMCs at a ratio of 1:10 (MSCs: PBMCs) for 5 days.

Conclusions
All PBMCs were then collected for assessment by ow cytometry to determine the positive percentage of CFSE-diluted cells (gated) to evaluate proliferation. AS-MSCs inhibited the proliferation of PBMCs, and this effect was strengthened by the activation of TLR4. C: The gene expression of cytokines and chemokines in AS-MSCs after LPS stimulation was detected by qRT-PCR. The symbol "*" represents P < 0.05.