IL1β Induces Mesenchymal Stem Cells Migration and Leucocyte Chemotaxis Through NF-κB

Mesenchymal stem cells are often transplanted into inflammatory environments where they are able to survive and modulate host immune responses through a poorly understood mechanism. In this paper we analyzed the responses of MSC to IL-1β: a representative inflammatory mediator. Microarray analysis of MSC treated with IL-1β revealed that this cytokine activateds a set of genes related to biological processes such as cell survival, cell migration, cell adhesion, chemokine production, induction of angiogenesis and modulation of the immune response. Further more detailed analysis by real-time PCR and functional assays revealed that IL-1β mainly increaseds the production of chemokines such as CCL5, CCL20, CXCL1, CXCL3, CXCL5, CXCL6, CXCL10, CXCL11 and CX3CL1, interleukins IL-6, IL-8, IL23A, IL32, Toll-like receptors TLR2, TLR4, CLDN1, metalloproteins MMP1 and MMP3, growth factors CSF2 and TNF-α, together with adhesion molecules ICAM1 and ICAM4. Functional analysis of MSC proliferation, migration and adhesion to extracellular matrix components revealed that IL-1β did not affect proliferation but also served to induce the secretion of trophic factors and adhesion to ECM components such as collagen and laminin. IL-1β treatment enhanced the ability of MSC to recruit monocytes and granulocytes in vitro. Blockade of NF-κβ transcription factor activation with IκB kinase beta (IKKβ) shRNA impaired MSC migration, adhesion and leucocyte recruitment, induced by IL-1β demonstrating that NF-κB pathway is an important downstream regulator of these responses. These findings are relevant to understanding the biological responses of MSC to inflammatory environments. Electronic supplementary material The online version of this article (doi:10.1007/s12015-012-9364-9) contains supplementary material, which is available to authorized users.


Introduction
Mesenchymal stem cells have become a therapeutic option for several pathologies like myocardial infarction, osteogenesis Supported by grants from the Instituto de Salud Carlos III for the Regenerative Medicine Program to Centro de Investigación Principe Felipe, from the FIS (PI07/784, CP08/80) and from Kutxa.

Electronic supplementary material
The online version of this article (doi:10.1007/s12015-012-9364-9) contains supplementary material, which is available to authorized users. imperfecta, graft versus host disease and wound healing [1][2][3][4]. As a part of the cell therapy, MSC are often transplanted in ischemic, apoptotic and/or inflammatory environments where cells survive and promote tissue regeneration by mechanisms that remain poorly understood. These cells are immunoprivileged, and in most of pathologies the induced potential benefits are related to paracrine activity mediated by their ability to survive in ischemic and inflammatory environments [5][6][7]. Despite their therapeutic potential initial, clinical results have been disappointing due to reported low benefits. It is believed that in adequate doses, low engraftment and poor survival are responsible for these results. We and others reported that intramyocardial MSC transplantation recruits a number of inflammatory cells that contribute to the healing of the infarct [8,9]. Transplanted cells are consistently exposed to tissue signals, immune cells and mediators that could influence their behaviour. Since successful application of stem cell approaches will depend on the microenvironment of the recipient tissue, we have sought to investigate the response of MSC to an inflammatory environment. Previous reports showed that proinflammatory cytokines were able to increased migration of human MSC to many chemotactic factors [10], to induce MSC to produce chemokines [11] and to stimulate MSC to differentiate into neural phenotype [12]. Following this rationale we cultured MSC in the presence of inflammatory mediators and analyzed biological responses implicated in proliferation, survival, adhesion and migration that could aid to predict their response in these environments. We focused our studies in IL-1β as a prototypical inflammatory mediator and the results showed that this cytokines promotes specific biological processes in MSC in part due to activation of the transcription factor NF-κB (Nuclear Factor KAPPA-light-chain-enhancer of activated B cells).

Cell Cycle Assay
To analyze the effect of IL-1β in cell cycle, 10 6 cells were harvested, fixed with 70% EtOH and kept at −20°C until use. Fixed cells were centrifugated and resuspended in 50 μg/mL propidium bromide (Sigma-Aldrich) and analyzed by flow cytometry (488 nm excitation, 625 nm emission).

MSC Migration Assay
To study trophism induced in MSC by IL-1β, cells were seeded in basal medium (DMEM with 0.5% FBS) at 10,000 cells/cm 2 in the top chamber of an 8 μm-pore migration transwell (BD Falcon, Bedford, MA, htpp://www.bd.com). After overnight culture, 25 ng/mL of IL-1β was added to the bottom chamber and cells were fixed with 2% paraformaldehide (Panreac Química, Castellar del Vallés, Spain), SDF-1α (20 ng/mL) and 10% FBS were used as positive controls. Briefly, after 4 h non migrated cells were removed from the upper side of the membrane using a cotton bud to remove non migrating cells, the membrane was cut and placed in a glass slide with the bottom side upwards and before staining with 4´,6 diamidino-2 phenyilindole (DAPI) (Sigma-Aldrich). All assays were performed in duplicated wells and repeated three times. Migrated cells were counted as fold increase relative to passive MSC cell migration in untreated wells.

Leucocyte Migration Assay
To determine the nature of human leucocytes that could be recruited in response to paracrine factors secreted by MSC, we established co-culture of MSC and pheripheral blood leucocytes (PBLs) using a transwell culture system (BD Falcon). MSC (10,000 cells/cm 2 ) seeded in the lower chamber of the transwells were stimulated or not with IL-1β for 2 h. After extensive washes with PBS, wells were filled with fresh medium and human PBLs from buffy coats (100,000 cells) were seeded in the upper chamber. Migrated cells through 8 μm-pore size membranes were counted after 5 h of coculture. Cells were fixed as described above and leucocyte populations were quantified in hematoxilin stained preparation by morphologic counting. All studies were performed in a blinded fashion.

Reverse Transcription and Real-Time Quantitative PCR
MSC incubated in different conditions were washed with PBS. RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA; htpp://www.invitrogen.com) and purified with RNeasy Plus Mini Kit (Qiagen, Dusseldorf, Germany; htpp://www.qiagen.com). RNA samples were quantified by spectrometry (NanoDrop ND-1000, NanoDrop Technologies, Wilmington, DE; htpp://www.nanodrop.com) and integrity was assessed by agarose gel electrophoresis and the absorbance ratio 260/280 nm. cDNA was obtained by retrotranscriptase reverse transcription using M-MLV Reverse Transcriptase (Invitrogen) from total RNA (1 μg). Primers were designed using the Primer-Blast online tool ( Table 1). The Ct of each PCR in a reference human MSC cDNA sample (dilution 1/10) is expressed as mean ± standard deviation (SD) of three independent PCR experiments. Real-time PCR was performed using convenient primers and SYBR Green I [1X LightCycler 480 SYBR Green I Master (Roche Molecular Biochemical, Mannheim, Germany; htpp://www.roche.com)]. Plates were run in a real-time thermal cycler (LightCycler 480 Instrument; Roche Diagnostics, Mannhein, Germany, htpp://www.roche. com) following manufacturer´s instructions. Real-time monitoring of the PCR reaction was performed with the LightCycler 480 Software 1.5, as well as the quantification of the products in the exponential phase of the amplification. Relative expression levels were calculated with the Relative

Microarray Data Analysis
Signal was standardized across arrays using quantile normalization [14]. Differential gene expression was carried out using the fold change. Gene set analysis was carried out for the Gene Ontology terms using FatiScan [15] from Babelomics web tool [16]. GO annotation for the genes in the microarray where taken from Ensembl 55 release (http://www.ensembl.org, Ensembl org, Hinxton, UK), allowing the visualization of functional categories within their biological context. Results were considered to be significant with a 2-fold induction.
The microarrays data of this study have been deposited in the Gene Expression Omnibus database under accession number GSE33755.

Statistical Analysis
Data are expressed as mean ± SD. Comparisons between experimental groups were done with unpaired and paired two-samples t-test using the SPSS software (SPSS, Chicago, IL http://www.spss.com). Differences were considered statistically significant at P<0.05.

Global Transcriptome Profiling of MSC Cultured with IL-1β
To test the effect of IL-1β on MSC, cells were cultured with or without 25 ng/mL of IL-1β for 24 h. Gene expression changes induced by the pro-inflammatory cytokine were evaluated by microarray analysis. Further bioinformatics analysis using Babelomics software (http://www.babelomics4.org) was performed to classify genes by function using the Gene Ontology (GO) scheme, revealing the main families of genes affected by the treatment. Growth in IL-1β resulted in activation of genes associated to very specific GO categories. In particular, we identified alterations in the expression of genes implicated in the following biological processes: i) response to wounding, ii) immune and inflammatory response, iii) defense response, iv) chemotaxis, v) locomotory behaviour, vi) regulation of cell proliferation, vii) leukocyte chemotaxix, viii) I-kappaB kinase/NF-kappaB cascade, ix) negative regulation of apoptosis, x) blood coagulation, and xi) cell adhesion ( Table 2). Fold changes of up-regulated genes (negative values) from enriched biological processes in MSC treated with IL-1β (MSC-IL1β) are indicated (Supplemental Table 1).

IL-1β Increases Expression of Multiple Chemokines and Growth Factors in MSC
After bioinformatic analysis, highly up-regulated genes related with these biological processes were further assayed by real- time PCR (Fig. 1, Table 3). Chemokines are small molecules that direct the migration of immune cells via chemokinechemokine receptor interactions. Based on their genetic organization and the position of two highly conserved cysteine residues at the N-terminus, chemokines can be divided into four subgroups, the CC, CXC, C, and CX 3 C families [17]. Among CC chemokines, CCL5 and CCL20 were the most up-regulated in response to IL-1β treatment (312±27 and 187±15 fold, respectively). CXCL1, CXCL3 and CX 3 CL1 were also highly expressed after IL-1β treatment. CXCL10, CXCL11 and ELF3 were expressed de novo upon stimulation of MSC with IL-1β (Table 3).
Regarding the cell adhesion molecules, the most important families include the intercellular adhesion molecules (ICAMs), the vascular adhesion molecules (VCAMs), cadherins, and selectins. Among them, IL-1β treatment increased expression of the integrin binding sialoprotein (IBSP), ICAM1, ICAM4, integrin beta 3 platelet glycoprotein IIIa (ITGB3), TCAM1P and VCAM1 as detected by real-time PCR ( Fig. 1 and Table 3). Other adhesion molecules also showed a significant fold detected by microarray analysis (Supplemental Table 1). Treatment with IL-1β influenced the secretion of interleukins and growth factors. The highest differences in fold change were found in TNF-α, followed by IL-8 and CSF2 levels ( Fig. 1 and Table 3). Whereas TNFα is a master inflammatory cytokine implicated in many biological process, IL-8 and CSF2 have more restricted biological activities. Indeed, IL-8 has been predominantly associated to chemotaxis of neutrophils [18] Chemokines (CXCLX)  whereas CSF2 is implicated in monocytic differentiation [19].

IL-1β induced tanscriptional levels (Normalized values)
Other biological processes activated in response to IL-1β were related to host defence and immune response. Microarray analysis and real time PCR experiments showed up-regulation of several Toll-like receptors (TLRs), claudins and NOD proteins. These molecules are implicated in the innate immune response to microbial infection. However, recent reports have revealed that these molecule also modulates biological processes in MSC such as differentiation, migration and immunomodulatory responses [20,21].

IL-1β Activates the NF-κB Pathway and do not Induce MSC Proliferation
We next analyzed the effect of IL-1β on BM-MSC signal transduction and cell proliferation. IL-1β promoted phosphorylation of NF-κB, but not PI3K/AKT and ERK1/2 pathways (Fig. 2a), as reported for other cell types [22]. However, in correlation with the result of the microarray analysis (Table 2), IL-1β did not induce significant cell proliferation as assessed by MTT assay (Fig. 2b). These results were further confirmed by cell cycle analysis using flow cytometry (Fig. 2c)

IL-1β Induced Migration and Adhesion of MSC is Mainly Activated Through NF-κB Signaling
We and others have previously described that MSC are able to migrate in vivo to ischemic and pro-inflammatory environments [8,23,24] and it is believed that this behaviour may underlie the ability of these cells to accelerate wound healing. Migration of MSC towards cytokines, chemokines and growth factors has also been explored in vitro [10]. To test if IL-1β was able to increase migratory ability in MSC, we cultivated MSC in the upper chamber of a transwell and stimulated migration by adding SDF-1α, IL-1β or 10% FBS in the lower chamber (Fig. 3a). A negative control of for migration was achieved using the same proportion of fetal bovine serum (0.5% FBS) in the upper and lower chamber. SDF-1α was used since it is a well-known trophic factor for MSC implicated in homing to ischemic areas [24], and 10% FBS was used as positive control since it is a rich source in cytokines and growth factors. Surprisingly, the migratory response of MSCs to IL-1β was in fact more pronounced than it was to to SDF-1α (1.68±0.21 fold increase versus 1.35±0.16), indicating a strong promigratory role for IL-1β Maximum migration was achieved towards FBS gradient (1.87 ± 0.12 fold increase). Similar levels of cell migration were observed when TNF-α or IL-8 were used as trophic factors (not shown), indicating that multiple inflammatory mediators can exert trophic effects on MSC as reported [24]. We next wanted to investigate whether the signaling pathways induced by IL-1β could be directly linked to MSC migration towards trophic factors. NF-κB transcription factors play an important role in the balance between cell survival and apoptosis and are involved in the regulation of cell proliferation and differentiation of various cell types [25]. IKKβ phosphorylates IκB molecules, the inhibitors of NF-κB, leading to ubiquitination and proteasome degradation of the inhibitors, and hence release and activation of NF-κB [26]. NF-κB has previously been described as the main transcription factor activated in many pro-inflammatory responses [27]. In these context, regulation of NF-κB cascade members was observed among the biological processes most positively affected by IL-1β treatment ( Table 2) and phosphorylation of NF-κB was induced on MSC after IL-1β treatment (Fig. 2). Thus, we sought to evaluate the role of NF-κB signaling in the biological responses of MSC in response to IL-1β. For this purporse, we constructed a vector containing shRNA targeting IKKβ that was lentiviraly transduced in MSC. We then evaluated the migratory response to IL-1β, SDF-1α and FBS. As shown in Fig. 3a, treatment with IKKβ shRNA reduced trophic response of MSC towards each of the 3 trophic factors assayed. An increase in the basal response of IKKβ transduced cells of 1.05±0.11 fold was observed, and in response to trophic factors this was increased by 1.21±0.11 towards SDF-1α, 1.45±0.06 towards IL-1β, and 1.58 ± 0.07 towards 10% FBS, strongly suggesting that NF-κB signaling pathway plays a major role in MSC trophism.
Migration and invasiveness of adherent cells is in part mediated by changes in the affinity of cells to particular ECM components (ECM). To test whether IL-1β had an effect on MSC cell adhesion, we measured the adhesion of MSC to the main components of ECM. The results showed that IL-1β treatment increased the adhesion to collagen (3.03±0.29 fold), fibronectin (1.75±0.11 fold) and laminin (2.79±0.15 fold) (Fig. 4b). In similar way to migration experiments, adhesion induced by IL1β treatment to collagen (1.75±0.15 fold), fibronectin (1.20±0.05 fold) and laminin (1.32±0.07 fold) was impaired in IKKβ-MSC. The fact that IKKβ expression only affected the adhesion induced by IL-1β but not the basal levels of adhesion to extracellular matrix components indicates that IKKβ blocks specifically the mechanisms induced by this cytokine, confirming the importance of NF-κB signaling pathway in the IL-1β mediated biological processes.

Il-1β Treatment of MSC Increases Recruitment of Leucocytes In Vitro
MSC have been shown to recruit inflammatory cells such as neutrophils, eosinophils, macrophages and to suppress proliferation of cytotoxic and helper T cells through the release of soluble factors such as HGF and TGF-β [11,[28][29][30]. Moreover, infusion of MSC into myocardium and other tissues is accompanied by marked, paracrine mediated leucocytic infiltration [4,8]. In order to test whether IL-1β treatment had a similar impact in MSC leucocyte recruitment, we cultured control or IL-1β treated MSCs, in a transwell system and measured the number and the type of leucocytes that migrate through a the 8 μm pores of the membrane.

Discussion
MSC have been used to treat a wide variety of diseases. Whilst the contribution of differentiation/transdifferentiation to tissue repair, are often minimal, other positive angiogenic and immunomodulatory effects are exerted by MSC in ischemic, apoptotic and pro-inflammatory environments [6]. IL-1β is produced in different tissues, not only as a response to pathogens, but also as a danger signals in pathologies such as acute myocardial infarction [31], type 2 diabetes [32], neural disorders [33]. In this study we wanted to investigate the response of MSC to proinflammatory stimuli in terms of survival, proliferation and induced paracrine factors. Thus, we treated MSC with IL-1β and used microarray to infer the biological response, firstly by gene function and later by direct gene set with known functional outcomes. A range of biological responses were activated in response to IL-1β, but perhaps, the most prominent was the potent stimulation of secretion of chemokines and growth factors that in turn were able to increase migration and adhesion of MSC and to regulate recruitment of monocytes and granulocytes. It is known that members of the CC family target primarily monocytes and T cells, whereas CXC chemokines affect mainly neutrophils. It has been previously reported that the existence of different monocyte subsets expressing different chemokine receptors display distinct migratory and functional properties. Interestingly, the profile by MSCs in response to chemokines secreted IL-1β, was enriched in CCL5, CCL20 and CX3CL1, that could specifically attract not only neutrophils and monocytes, but also monocytes expressing CCR5, CCR6 and high levels of CX3CR1 [34]. Although leukocyte chemotaxis and lymphocyte development are the main functional properties of chemokines, they posses other biological activities like regulation of angiogenesis, control of cell proliferation and alteration of the expression of adhesion molecules. Indeed, the structural ERL domain present in several members of the CXC chemokine family determines their angiogenic potential [35] and the induced chemokquines CXCL1, CXCL3, and CXCL8 (IL-8) contain this motif. In the same context, CXCL10 is considered a "stop signal" that limits expansion of the fibrotic reaction triggered by TGFβ, FGF, and VEGF during myocardial healing [31]. The high levels of activation of this chemokine in MSC (Table 3) could account for the potent ability of these cells to control adverse remodeling during myocardial healing [8,36,37].
Claudins are transmembrane proteins found in tight juntions that participate not only in regulating tissue barrier function and permeability but also in cell motility, adhesion and migration [38]. Claudins (CLDN1 and CLDN14) were up-regulated in MSC after IL-1β treatment. A similar response has been reported in airway smooth muscle cells in response to IL-1β and TNFα [39], indicating similar activation pathways. It has been described that TLR signalling is linked to NF-κB and MAPK signalling pathways, and that this induction mediates the secretion chemokines and regulates immunosuppressive activity and recruitment of innate immune cells [21,40,41]. TLR2 and TLR4 were upregulated in response to IL-1β. Similar effect had been previously described after stimulation with LPS of MSC from human parotid glands [42].
We also found differences between the activation pattern of MSC in response to different inflammatory mediators. Whereas TNFα increased preferentially CCL2 (MCP-1), CCL5 (RANTES), CXCL1, CXCL5, CXCL8, CXCL10 and CCL11 [10], we demonstrate here that IL-1β increases preferentially CCL3, CCL5, CCL20, CXCL1,CXCL3, CXCL10 and CXCL11. Thus, modulation of MSC biological responses is closely associated with culture conditions and the presence of immune mediators influence MSC proliferation and multipotency. In this context, culture protocols with milieu capable of MSC expansion while preserving chromosome stability have been developed [43] In summary, our findings show that IL-1β increases migration and adhesion of MSC and promotes leucocyte chemotaxis through MSC secretion of soluble factors. As described in other cell types [44], IL-1β activates NF-κB resultings in transcriptional activation of a wide variety of genes such inflammatory mediators, adhesion molecules, growth factor or immune response mediator. Since some of these molecules are chemotactic for inflammatory leukocytes, like monocytes and neutrophils, these paracrine factors could facilitate infiltration of immune cells for tissue repair when MSC are transplanted into injured tissues.
Taken together, these findings shed light on MSC behaviour in inflammatory environments and suggest that inflammatory mediators like IL-1β induce a response in MSC that could trigger paracrine actions in vivo.