Lipopolysaccharide activates Toll-like receptor 4 (TLR4)-mediated NF-κB signaling pathway and proinflammatory response in human pericytes

Abstract

Pericytes and mesenchymal stem cells (MSCs) are ontogenically related, and in fact, no significant phenotypic differences could be observed by flow cytometry. Transcriptome analysis of human pericytes and MSCs revealed that 43 genes were up-regulated more than 10-fold in pericytes compared with MSCs. Identification of Toll-like receptor 4 (TLR4) as one of the most abundant RNA species in pericytes with respect to MSCs and confirmation of TLR4 expression on the cell surface led us to obtain a comprehensive overview of the expression program of lipopolysaccharide (LPS)-stimulated pericytes. Transcriptional profiling of LPS-treated cells revealed that 22 genes were up-regulated more than 5-fold. Of them, 10 genes encoded chemokines and cytokines (CXCL10, CCL20, IL8, CXCL1, IL6, CCL2, IL1B, CXCL2, IL1A, and CXCL6), and three genes encoded adhesion molecules (ICAM1, VCAM1, and SELE). LPS induced nuclear translocation of the transcription factor NF-κB in stimulated pericytes. Moreover, inhibition of NF-κB activation by SC-514 blocked LPS-induced up-regulation of a subset of chemokine genes, confirming the key role of NF-κB in LPS signaling in pericytes. At the protein level, we assessed the secretion of the proinflammatory cytokines and chemokines IL-6, IL-8, CXCL1, CXCL2, CXCL3, and CCL2 not only after LPS treatment but also in HMGB1-stimulated pericytes. Up-regulation of the adhesion molecules ICAM-1 and VCAM-1 resulted in an increased adhesion of peripheral blood leukocytes to an LPS-treated pericyte monolayer. The role of pericytes in the inflammatory context has been scarcely addressed; according to these results, pericytes should be considered as active players in the inflammatory cascade with potential physiopathological implications.

Keywords: Angiogenesis; Chemokines; Cytokine; HMGB1; Inflammation; Lipopolysaccharide (LPS); NF-κB; Pericyte; TLR4.

FIGURE 1.

Comparative phenotypic analysis of HBVPs and MSCs. Cells were labeled with antibodies against the indicated antigens and analyzed by flow cytometry. Isotype-matched antibodies were used as control (in solid gray). Numbers indicate the percentage of positive cells together with the mean fluorescence intensity (in parentheses).

FIGURE 2.

Cell surface expression of TLR4 detected by flow cytometry in HBVPs and MSCs stimulated or not with LPS. Filled histograms denote background fluorescence; line histograms denote TLR4 staining.

FIGURE 3.

Expression of TLR4 signaling molecules in HBVPs. Immunoblot analysis of MyD88, MD-2, and CD14 is shown. β-Actin was used as a loading control.

FIGURE 4.

qRT-PCR analysis of genes selected from the microarray profile in LPS-stimulated HBVPs and effect of HMGB1 on gene expression. A, expression levels of five selected genes were evaluated by qRT-PCR to validate microarray data in HBVPs treated with 1 μg/ml LPS for 4 h. B, analysis of IL6 and IL8 levels by qRT-PCR in HBVPs stimulated with different concentrations of HMGB1 for 4 h.***, p < 0.001. Error bars represent S.D.

FIGURE 5.

Secretion of cytokines and chemokines by HBVPs and MSCs after LPS and HMGB1 stimulation. A, semiquantitative detection of cytokines and chemokines in the conditioned media of HBVPs and MSCs treated with 1 μg/ml LPS for 4 h. B, cytokine quantification in the conditioned medium of LPS-stimulated HBVPs. C, semiquantitative detection of cytokines and chemokines in the conditioned media of HBVPs and MSCs treated with 10 μg/ml HMGB1 for 4 h. Data are representative of three independent experiments (mean ± S.D. of triplicates). *, p < 0.05; ***, p < 0.001. Error bars represent S.D.

FIGURE 6.

LPS mediates activation of the NF-κB signaling pathway in HBVPs but not phosphorylation of p38 MAPK. A, NF-κB p65 is detected in the cytoplasm of unstimulated cells (arrowheads show non-stained nuclei). Nuclear translocation of NF-κB p65 takes place after LPS stimulation for 90 min (arrowheads indicate stained nuclei). Original magnification, ×400. B, suppression of LPS-promoted CXCL1, -2, and -3 gene expression by an NF-κB inhibitor. HBVPs were incubated with different doses of the NF-κB inhibitor SC-514 for 30 min prior to LPS stimulation for 90 min. RNA was isolated and analyzed by qRT-PCR. *, p < 0.05; **, p < 0.005. C, LPS-promoted CXCL1, -2, and -3 up-regulation is not disrupted in HBVPs treated with different mitogen-activated protein kinase inhibitors, SP600125 (JNK1 and -2 inhibitor), PD98059 (MEK-1 inhibitor), and SB203580 (p38 inhibitor), prior to LPS stimulation. D, LPS treatment does not alter the expression level or phosphorylation status of p38 MAPK as assessed by Western blot at different time points. As a positive control, cells were treated with 10 μm anisomycin to induce p38 MAPK activation. Results shown in A and D represent those obtained in at least three independent experiments. Error bars represent S.D.

FIGURE 7.

Expression of ICAM-1 and VCAM-1 on the surface of LPS-treated HBVPs and MSCs. A, ICAM-1 and VCAM-1 expression was analyzed by flow cytometry at different time points after LPS addition. Isotype-matched antibodies were used as control (in solid gray). B, bar graphs represent the mean fluorescence intensities (MFI) of each marker and time point. One representative result from three independent experiments is shown.

FIGURE 8.

PBL adhesion to HBVPs treated with LPS. A, fluorescently labeled PBLs were added onto a confluent monolayer of LPS-treated HBVPs and washed after 2 h. Original magnification, ×200. B, quantification of adhered cells. Values correspond to the arithmetic mean ± S.D. of three different fields belonging to a representative experiment. *, p < 0.05; **, p < 0.005. Error bars represent S.D.