Overexpression of hypoxia-inducible factor 1 alpha improves immunomodulation by dental mesenchymal stem cells

Abstract

Background: Human dental mesenchymal stem cells (MSCs) are considered as highly accessible and attractive MSCs for use in regenerative medicine, yet some of their features are not as well characterized as other MSCs. Hypoxia-preconditioning and hypoxia-inducible factor 1 (HIF-1) alpha overexpression significantly improves MSC therapeutics, but the mechanisms involved are not fully understood. In the present study, we characterize immunomodulatory properties of dental MSCs and determine changes in their ability to modulate adaptive and innate immune populations after HIF-1 alpha overexpression.

Methods: Human dental MSCs were stably transduced with green fluorescent protein (GFP-MSCs) or GFP-HIF-1 alpha lentivirus vectors (HIF-MSCs). A hypoxic-like metabolic profile was confirmed by mitochondrial and glycolysis stress test. Capacity of HIF-MSCs to modulate T-cell activation, dendritic cell differentiation, monocyte migration, and polarizations towards macrophages and natural killer (NK) cell lytic activity was assessed by a number of functional assays in co-cultures. The expression of relevant factors were determined by polymerase chain reaction (PCR) analysis and enzyme-linked immunosorbent assay (ELISA).

Results: While HIF-1 alpha overexpression did not modify the inhibition of T-cell activation by MSCs, HIF-MSCs impaired dendritic cell differentiation more efficiently. In addition, HIF-MSCs showed a tendency to induce higher attraction of monocytes, which differentiate into suppressor macrophages, and exhibited enhanced resistance to NK cell-mediated lysis, which supports the improved therapeutic capacity of HIF-MSCs. HIF-MSCs also displayed a pro-angiogenic profile characterized by increased expression of CXCL12/SDF1 and CCL5/RANTES and complete loss of CXCL10/IP10 transcription.

Conclusions: Immunomodulation and expression of trophic factors by dental MSCs make them perfect candidates for cell therapy. Overexpression of HIF-1 alpha enhances these features and increases their resistance to allogenic NK cell lysis and, hence, their potential in vivo lifespan. Our results further support the use of HIF-1 alpha-expressing dental MSCs for cell therapy in tissue injury and immune disorders.

Keywords: Cell therapy; Dental pulp; Hypoxia-inducible factor 1 alpha subunit (HIF-1 alpha); Immunomodulation; Mesenchymal stem cells (MSCs).

Fig. 1

Overexpression of HIF-1 alpha and mitochondrial stress test in MSCs. a Representative Western blot of hypoxia-inducible factor-1 (HIF-1) alpha in mesenchymal stem cells (MSCs) and HIF-MSCs. Loading control was performed with tubulin and levels of HIF-1 alpha protein expression were quantified by densitometry (right panel). b Expression of the HIF-1 alpha mRNA determined by quantitative PCR. GNB2L1 was used as endogenous control. Date represent expression in HIF-MSCs relative to MSCs control (fold change; FC). Quantification of four independent experiments; data are represented as mean ± SE (Student t test, *p ≤ 0.05). c Oxygen comsumption rate (OCR) response to oligomycin (Oligo.; 1 μM), carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP; 1 μM), and antimycin A/rotenone (Ant.A/Rot.; both 1 μM) injections. HIF-MSCs (gray line) showed a lower OCR than MSCs (black line). d Mitochondrial substrate utilization in HIF-MSCs (gray) and MSCs (black) measured in terms of basal respiration, ATP production, non-mitochondrial respiration, maximal respiration, proton leak, and spare respiratory capacity. Data were normalized against the total amount of protein. Data are represented as mean ± SE of six independent experiments (Student t test, *p ≤ 0.05, **p ≤ 0.01). GFP green fluorescent protein

Fig. 2

HIF-1 alpha overexpression does not modify the ability of MSCs to inhibit T-cell activation. T cell-enriched peripheral blood cells were stained with carboxyfluoroscein succinimidyl ester (CFSE) and activated with anti-CD3/anti-CD28 monoclonal antibodies in the presence or absence of mesenchymal stem cells (MSCs; light grey) and hypoxia-inducible factor (HIF)-MSCs (dark grey). T cells (TC) were cultured in media alone as a negative control. After 4 days, expression of CD25 and proliferation of T-cell subsets was determined by flow cytometry. A representative experiment (a) and the mean ± SD of three independent experiments (b) are shown. T cells were gated according to forward/side scatter characteristics and expression of CD3, CD4, and CD8. c Supernatants of cultures were collected after 4 days and assayed for interferon (IFN)-gamma production. Data are presented as mean ± SD of three independent experiments. One-way ANOVA, *p ≤ 0.05, GFP-MSCs and HIF-MSCs versus no MSCs. GFP green fluorescent protein

Fig. 3

Inhibition of dendritic cell differentiation by dental MSCs. Monocytes were differentiated towards dendritic cells with rhGM-CSF and rhIL-4 alone (Mo) or in the presence of control green fluorescent protein (GFP)-mesenchymal stem cells (MSCs; light grey) and hypoxia-inducible factor (HIF)-MSCs (dark grey). After 7 days, cells were harvested, and the expression of CD14 and CD1a was analyzed by flow cytometry. A representative experiment (a) and mean ± SD of four independent experiments (b) are shown. MSCs were excluded from the analysis by CD90 expression. One-way ANOVA, *p ≤ 0.05, GFP-MSCs and HIF-MSCs versus no MSCs

Fig. 4

Monocyte differentiation and attraction in the presence of dental MSCs. Monocytes alone (Mo) or in the presence of green fluorescent protein (GFP)-mesenchymal stem cells (MSCs; light grey) and hypoxia-inducible factor (HIF)-MSCs (dark grey) were cultured with rhGM-CSF to induce macrophage differentiation. After 7 days, the expression of CD14 and CD163 was determined by flow cytometry. A representative experiment (a) and mean ± SD of three independent experiments (b) are shown. MSCs were excluded from the analysis by CD90 expression. c After 7 days of culture, LPS was added for 24 h and supernatants were assayed for tumor necrosis factor (TNF) alpha and interleukin (IL)-10 production. Data are presented as mean ± SD of three independent experiments. One-way ANOVA, *p ≤ 0.05, GFP-MSCs and HIF-MSCs versus no MSCs. d, e MSCs were cultured until 80% confluence, then fresh medium alone (Med) or medium supplemented with IFN-gamma (IFNγ) was added. After 15 h, cells and supernatants were harvested. Transcript (d) and protein levels (e) for CCL2 were determined by quantitative PCR and CBA, respectively. GNB2L1 was used as endogenous control. Mean ± SD of four samples pooled from two independent experiments is shown. Student t test, *p ≤ 0.05, MSCs vs HIF-MSCs). f Transwell (8 μm) cultures were performed as stated in the Methods section. After 14 h, migrated cells (present at the lower chamber) were collected and stained for CD14. MSCs were excluded from the analysis by CD90 expression and monocytes were gated according to forward/side scatter characteristics and expression of CD14, and counted in a FACSCalibur flow cytometer

Fig. 5

HIF-1 overexpression in MSCs confers resistance to NK cell-mediated lysis. Natural killer (NK) cells were cultured in media alone (black bars) or with rhIL-12 and rhIL-15 in the absence (white bars) or presence of green fluorescent protein (GFP)-mesenchymal stem cells (MSCs; light grey) and hypoxia-inducible factor (HIF)-MSCs (dark grey). After 36 h of culture, expression of CD107a (a), intracellular interferon (IFN)-gamma (c) and the indicated molecules (d) was analyzed in NK cells by flow cytometry. MSCs were excluded from the analysis by CD90 expression. b MSC-NK cell co-cultures were performed at the indicated MSC:NK cell ratios in presence of rhIL-12 and rhIL-15. After 36 h late apoptosis of MSCs was determined by CD90 expression and propidium iodide (PI) incorporation. A representative experiment (b and c) and mean ± SD of three independent experiments (a and d) are shown. One-way ANOVA, *p ≤ 0.05, GFP-MSCs and HIF-MSCs versus no MSCs. e, f MSCs were cultured until 80% confluence was reached, and then culture medium was replaced by fresh medium alone (e) or supplemented with IFN-gamma (f). After 15 h, cells were harvested and expression of the indicated genes was determined by qPCR. GNB2L1 was used as endogenous control. Bars represent expression in HIF-MSCs relative to GFP-MSC controls. Mean ± SD of six to eight samples pooled from at least three independent experiments is shown. Student t test, ^# p ≤ 0.05, ^## p ≤ 0.005, GFP-MSCs versus HIF-MSCs. IL interleukin

Fig. 6

Immunomodulatory and pro-angiogenic profile induced by HIF-1 alpha expression in MSCs. Mesenchymal stem cells (MSCs) were cultured until 80% confluence was reached, and then culture medium was replaced by fresh medium alone (Basal) or supplemented with IFN-gamma (IFNγ). After 15 h, cells were harvested and expression of the indicated genes was determined by qPCR. GNB2L1 was used as endogenous control. a, c Expression of the indicated genes in control green fluorescent protein (GFP)-MSCs. Note that a logarithmic scale is used. b, d Expression in hypoxia-inducible factor (HIF)-MSCs relative to MSC controls. Mean ± SD of six to eight samples pooled from at least three independent experiments is shown. Student t test, ^# p ≤ 0.05, MSCs vs HIF-MSCs