Inhibition of the Differentiation of Monocyte-Derived Dendritic Cells by Human Gingival Fibroblasts

Abstract

We investigated whether gingival fibroblasts (GFs) can modulate the differentiation and/or maturation of monocyte-derived dendritic cells (DCs) and analyzed soluble factors that may be involved in this immune modulation. Experiments were performed using human monocytes in co-culture with human GFs in Transwell® chambers or using monocyte cultures treated with conditioned media (CM) from GFs of four donors. The four CM and supernatants from cell culture were assayed by ELISA for cytokines involved in the differentiation of dendritic cells, such as IL-6, VEGF, TGFβ1, IL-13 and IL-10. The maturation of monocyte-derived DCs induced by LPS in presence of CM was also studied. Cell surface phenotype markers were analyzed by flow cytometry. In co-cultures, GFs inhibited the differentiation of monocyte-derived DCs and the strength of this blockade correlated with the GF/monocyte ratio. Conditioned media from GFs showed similar effects, suggesting the involvement of soluble factors produced by GFs. This inhibition was associated with a lower stimulatory activity in MLR of DCs generated with GFs or its CM. Neutralizing antibodies against IL-6 and VEGF significantly (P<0.05) inhibited the inhibitory effect of CM on the differentiation of monocytes-derived DCs and in a dose dependent manner. Our data suggest that IL-6 is the main factor responsible for the inhibition of DCs differentiation mediated by GFs but that VEGF is also involved and constitutes an additional mechanism.

Therapeutic Potential of Gingival Fibroblasts for Cutaneous Radiation Syndrome: Comparison to Bone Marrow-Mesenchymal Stem Cell Grafts

“…Physiological levels of these growth factors are critical for ensuring wound healing [40]. Recently, Séguier et al showed in vitro that the immunomodulatory effects of GFs involve VEGF secretion [21]. We showed here that VEGF, FGF7, and EGF were overexpressed earlier in GF-treated skin than in MSCtreated skin.…”

“…The mechanism of GF therapeutic effect on wound healing such as MSC therapy involved inflammatory modulation associated to a switch of macrophages to an M2-like phenotype and an ECM remodeling is reinforced by previous observations where GF secretion factors inhibited dendritic cell differentiation [21].…”

“…The study was approved by the local ethics committee (Comité de Protection des Personnes (CPP) ''Ile de France II'' (May 11, 2012), IRB registration: 00001072) and all subjects gave written informed consent. As previously reported [19,21], primary explant cultures were established in Dulbecco's modified Eagle's medium (DMEM) containing 5% human platelet lysate, penicillin (100 mg/mL), streptomycin (100 mg/mL), and amphotericin B (2 ng/mL) over 2-week periods. Monolayer cultures were maintained in 5% CO 2 and the cell culture medium was changed every 72 h. After 2 weeks, the GFs were trypsinized and single-cell suspensions were seeded at very low concentrations ( £ 100 cells/cm 2 ; medium, DMEM 10% of FBS with 50 mg/mL of ascorbic acid) after verification of the absence of cell aggregates to avoid false positives.…”

“…Gingival fibroblast (GF) culture, in which *3% of cells form colonies, expresses membrane markers similar to those of bone marrow-derived MSCs [18,19] and has, like them, immunomodulatory functions and the potential for multilineage differentiation [20,21]. The efficacy of GFs in wound healing was recently shown in a full-thickness skin defect [22].…”

Immune Contexture, Immunoscore, and Malignant Cell Molecular Subgroups for Prognostic and Theranostic Classifications of Cancers

“…As major producers of the extracellular matrix, they control the trafficking of T cells from the invasive margin (IM) to the tumor stroma (Salmon et al, 2012). Fibroblasts can directly inhibit TNF-α and IFN-γ-mediated antitumor immunity (Kraman et al, 2010), hamper DC maturation (Séguier et al, 2013), and inhibit T-cell proliferation (Bocelli-Tyndall et al, 2006;Haniffa et al, 2007;Jones, Horwood, Cope, & Dazzi, 2007). They have been shown to constitutively express the immune checkpoint ligand PD-L1 (Pinchuk et al, 2008), and this expression is upregulated upon IFN-γ stimulation (Pinchuk et al, 2008).…”