IL1β induces mesenchymal stem cells migration and leucocyte chemotaxis through NF-κB

Abstract

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 CX(3)CL1, 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.

Fig. 1

Quantification of gene transcription levels by qRT-PCR analysis in MSC cultures treated with IL-1β versus untreated cultures. Data are expressed as relative fold of control (normalized values) and represent the mean ± S.D. of three independent experiments

Fig. 2

a, Western blot analysis of kinase phosphorylation in MSC treated with IL-1β. Total cell lysates (30 μg) were separated by SDS-PAGE. Anti-GAPDH was used as loading control. b, MTT assay of MSC cultures treated with IL-1β (25 ng/mL) and IL-6 (20 ng/mL) for 24 h. Values are expressed in fold increase relative to control. c, Cell cycle analysis by flow cytometry of MSC cultures treated with IL-1β (25 ng/mL) for 24 h. (*P < 0.05)

Fig. 3

a, migration of MSC or MSC-IKKβ towards trophic factors SDF-1α (20 ng/mL), IL-1β (25 ng/mL) and 10% FBS. b, Adhesion of untreated MSC (black bars) and MSC-IKKβ (dashed bars) or treated with IL-1β (white and grey bars, respectively) to collagen, fibronectin and laminin. Data are represented as fold increase relative to MSC control. (*P < 0.05, **P < 0.01, ***P < 0.001 in both panels)

Fig. 4

Leucocyte migration assay of human PMNs using a transwell insert culture system towards different stimuli. Upper chamber were filled with PMNs and lower chambers were seeded with MSC (black bars) or MSC-IKKβ (white bars) treated (dashed and grey bars, respectively) or not with IL-1β. Migrated neutrophils, eosinophils, lymphocytes and monocytes are presented as a mean of three experiments ± S.D. (number of migrated cells/field). *P < 0.05, **P < 0.01, ***P < 0.001