Human Skin Culture as an Ex Vivo Model for Assessing the Fibrotic Effects of Insulin-Like Growth Factor Binding Proteins

Systemic sclerosis (SSc) is a connective tissue disease of unknown etiology. A hallmark of SSc is fibrosis of the skin and internal organs. We recently demonstrated increased expression of IGFBP-3 and IGFBP-5 in primary cultures of fibroblasts from the skin of patients with SSc. In vitro, IGFBP-3 and IGFBP-5 induced a fibrotic phenotype and IGFBP-5 triggered dermal fibrosis in mice. To assess the ability of IGFBPs to trigger fibrosis, we used an ex vivo human skin organ culture model. Our findings demonstrate that IGFBP-3 and IGFBP-5, but not IGFBP-4, increase dermal and collagen bundle thickness in human skin explants, resulting in substantial dermal fibrosis and thickening. These fibrotic effects were sustained for at least two weeks. Our findings demonstrate that human skin ex vivo is an appropriate model to assess the effects of fibrosis-inducing factors such as IGFBPs, and for evaluating the efficacy of inhibitors/therapies to halt the progression of fibrosis and potentially reverse it

Syndecan-2 is a novel target of insulin-like growth factor binding protein-3 and is over-expressed in fibrosis

Extracellular matrix deposition and tissue scarring characterize the process of fibrosis. Transforming growth factor beta (TGFβ) and Insulin-like growth factor binding protein-3 (IGFBP-3) have been implicated in the pathogenesis of fibrosis in various tissues by inducing mesenchymal cell proliferation and extracellular matrix deposition. We identified Syndecan-2 (SDC2) as a gene induced by TGFβ in an IGFBP-3-dependent manner. TGFβ induction of SDC2 mRNA and protein required IGFBP-3. IGFBP-3 independently induced production of SDC2 in primary fibroblasts. Using an ex-vivo model of human skin in organ culture expressing IGFBP-3, we demonstrate that IGFBP-3 induces SDC2 ex vivo in human tissue. We also identified Mitogen-activated protein kinase-interacting kinase (Mknk2) as a gene induced by IGFBP-3. IGFBP-3 triggered Mknk2 phosphorylation resulting in its activation. Mknk2 independently induced SDC2 in human skin. Since IGFBP-3 is over-expressed in fibrotic tissues, we examined SDC2 levels in skin and lung tissues of patients with systemic sclerosis (SSc) and lung tissues of patients with idiopathic pulmonary fibrosis (IPF). SDC2 levels were increased in fibrotic dermal and lung tissues of patients with SSc and in lung tissues of patients with IPF. This is the first report describing elevated levels of SDC2 in fibrosis. Increased SDC2 expression is due, at least in part, to the activity of two pro-fibrotic factors, TGFβ and IGFBP-3. © 2012 Ruiz et al

In Situ-Targeting of Dendritic Cells with Donor-Derived Apoptotic Cells Restrains Indirect Allorecognition and Ameliorates Allograft Vasculopathy

Chronic allograft vasculopathy (CAV) is an atheromatous-like lesion that affects vessels of transplanted organs. It is a component of chronic rejection that conventional immuno-suppression fails to prevent, and is a major cause of graft loss. Indirect allo-recognition through T cells and allo-Abs are critical during CAV pathogenesis. We tested whether the indirect allo-response and its impact on CAV is down-regulated by in situ-delivery of donor Ags to recipient's dendritic cells (DCs) in lymphoid organs in a pro-tolerogenic fashion, through administration of donor splenocytes undergoing early apoptosis. Following systemic injection, donor apoptotic cells were internalized by splenic CD11chi CD8α+ and CD8− DCs, but not by CD11cint plasmacytoid DCs. Those DCs that phagocytosed apoptotic cells in vivo remained quiescent, resisted ex vivo-maturation, and presented allo-Ag for up to 3 days. Administration of donor apoptotic splenocytes, unlike cells alive, (i) promoted deletion, FoxP3 expression and IL-10 secretion, and decreased IFN-γ-release in indirect pathway CD4 T cells; and (ii) reduced cross-priming of anti-donor CD8 T cells in vivo. Targeting recipient's DCs with donor apoptotic cells reduced significantly CAV in a fully-mismatched aortic allograft model. The effect was donor specific, dependent on the physical characteristics of the apoptotic cells, and was associated to down-regulation of the indirect type-1 T cell allo-response and secretion of allo-Abs, when compared to recipients treated with donor cells alive or necrotic. Down-regulation of indirect allo-recognition through in situ-delivery of donor-Ag to recipient's quiescent DCs constitutes a promising strategy to prevent/ameliorate indirect allorecognition and CAV

Apoptotic cell-based therapies against transplant rejection: role of recipient’s dendritic cells

One of the ultimate goals in transplantation is to develop novel therapeutic methods for induction of donor-specific tolerance to reduce the side effects caused by the generalized immunosuppression associated to the currently used pharmacologic regimens. Interaction or phagocytosis of cells in early apoptosis exerts potent anti-inflammatory and immunosuppressive effects on antigen (Ag)-presenting cells (APC) like dendritic cells (DC) and macrophages. This observation led to the idea that apoptotic cell-based therapies could be employed to deliver donor-Ag in combination with regulatory signals to recipient’s APC as therapeutic approach to restrain the anti-donor response. This review describes the multiple mechanisms by which apoptotic cells down-modulate the immuno-stimulatory and pro-inflammatory functions of DC and macrophages, and the role of the interaction between apoptotic cells and APC in self-tolerance and in apoptotic cell-based therapies to prevent/treat allograft rejection and graft-versus-host disease in murine experimental systems and in humans. It also explores the role that in vivo-generated apoptotic cells could have in the beneficial effects of extracorporeal photopheresis, donor-specific transfusion, and tolerogenic DC-based therapies in transplantation

Regulation of the systemic anti-donor response by donor apoptotic splenocytes.

<p>A) Assessment by IFN-γ ELISPOT assay of the indirect T cell response elicited in the spleens of B6 mice, naïve (control), or transplanted 60 days before with syngeneic (control) or BALB/c aortic grafts. Numbers indicate the number of spots. Each group included 4 mice. B) Detection by FACS of circulating alloAb in B6 recipients of syngeneic or BALB/c aortic grafts under different conditions and analyzed 60 days after surgery. Results are representative of 6 mice per group. Numbers indicate percentages of cells. Numbers in parenthesis represent mean fluorescence intensity. C) Evaluation of the direct T cell response by IFN-γ ELISPOT assay in the spleen of B6 mice, naïve (control) or 60 days after being transplanted with syngeneic (control) or BALB/c aortas, under different conditions. Each group included 4 mice per group. * p<0.001.</p

Administration of donor apoptotic cells regulates the indirect T cell responses.

<p>A) Allo-Ag presentation and ELISPOT assays of splenocytes from B6 mice reconstituted (or not, control) with 1H3.1 CD4 T cells and then injected i.v. with B6 (control) or BALB/c apoptotic splenocytes (alone or with agonistic CD40 mAb), or with BALB/c splenocytes alive. Fourteen days later, splenocytes from the host B6 mice were cultured alone or with the BALB/c peptide IEα_52–68 during 72 h for assessment of T cell proliferation by [³H]TdR incorporation, or for 36 h in ELISPOT plates for quantification of IFN-γ and IL-10 secretion. Each group included 6 mice. B) Analysis by FACS of the percentage of CD4 1H3.1 T cells (Thy1.1⁺) expressing FoxP3 in the spleen of host B6 mice (Thy1.2⁺) 14 days after receiving i.v. B6 (control) or BALB/c apoptotic splenocytes, or BALB/c splenocytes alive. Numbers indicate percentages of cells. One representative of 6 individual experiments is shown. * p<0.01. C) Assessment by in vivo killing assays of the Ag-specific lytic activity of B6 mice reconstituted with 2C CD8 T cells and treated (day 1) with 10⁷ MHC-I^−/− B6 apoptotic or alive splenocytes loaded (or not) with the H2K^b-retricted SYGL peptide. Mice were challenged (or not) on day 6 with SYGL plus IFA (i.p.). On day 9, mice were i.v. injected with a 1/1 mixture of CFSE^hi BALB/c×B6 (F1) (target) and CFSE^lo B6 (control) splenocytes. Five h later, the ratio of CFSE^hi vs. CFSE^lo cells in the spleen was determined by FACS. Numbers indicate percentages of cells. The specific lysis was calculated and is shown as the mean±SD of 3 mice per group.</p

Donor apoptotic cells are recognized by indirect CD4 T cells.

<p>A) Analysis by FACS of proliferation and phenotype of CFSE-labeled 1H3.1 CD4 T cells transferred into B6 mice that were then treated (i.v.) with BALB/c splenocytes, alive or apoptotic. As control, to promote 1H3.1 T cell activation/proliferation, a group of B6 mice was treated with BALB/c apoptotic cells plus agonistic CD40 mAb. Numbers in dot plots represent percentages of cells. Numbers in parenthesis indicate the mean fluorescence intensity of the left quadrants combined. One representative experiment out of 6 is shown. B) ELISPOT analysis for IFN-γ of splenocytes from B6 mice reconstituted (or not, control) with 1H3.1 CD4 T cells and then injected i.v. with B6 (control) or BALB/c apoptotic splenocytes (alone or with agonistic CD40 mAb), or with BALB/c splenocytes alive. Three days later, splenocytes from the host B6 mice were cultured for 36 h in ELISPOT plates alone, or with the BALB/c peptide IEα_52–68. Each group included 6 mice. C) Assessment by FACS of the percentages of CD4 1H3.1 T cells (Thy1.1⁺) in tissues of host B6 mice (Thy1.2⁺) previously reconstituted with 1H3.1 cells and then treated with B6 (control) or BALB/c apoptotic splenocytes (the latter alone or with agonistic CD40 mAb), or with BALB/c splenocytes alive. Results represented values pooled from 6 mice per group. * p<0.01.</p

Splenic DCs remain quiescent following interaction with donor apoptotic cells in vivo.

<p>A) B6 mice were left untreated or injected i.v. with PKH67-labeled BALB/c apoptotic splenocytes. Histograms show the expression of surface markers by splenic CD11c^hi DCs (i) with internalized PKH-67⁺ apoptotic cell fragments (thick line), (ii) without PKH-67⁺ fragments from the same mice (gray), and (iii) from control non-injected mice (dotted line). One representative out of 3 individual experiments is illustrated. B) Eighteen h after i.v. administration of PKH-67-labeled BALB/c apoptotic splenocytes in B6 mice, splenic CD11c^hi DCs with internalized PKH-67⁺ fragments (R2) or not (R1) were FACS-sorted and cultured in medium with GM-CSF and LPS. After 24 h, surface expression of MHC class-II Ag and CD86 was analyzed by flow cytometry in the FACS-sorted splenic DCs that had internalized PKH-67⁺ fragments (gray histograms) or not (thick line histograms), and compared to the phenotype of freshly-isolated splenic CD11c^hi DCs (dotted line histogram). One representative out of 3 experiments is shown. C) CFSE-labeled 1H3.1 CD4 T cells were adoptively transferred (i.v.) into B6 mice at different times after administration of BALB/c splenocytes (alive, apoptotic or necrotic) and proliferation of 1H3.1 T cells was evaluated based on CFSE dilution assessed by FACS, 3 days after T cell transference. Presentation of BALB/c allopeptides to splenic 1H3.1 CD4 T cells (based on CFSE-dilution) decreased drastically by 7 days. Numbers represent percentages of dividing cells. One representative experiment with 3 mice per group and time point is shown.</p