Giuseppina Pennesi

Bone marrow mesenchymal progenitor cells inhibit lymphocyte proliferation by activation of the programmed death 1 pathway.

Bone marrow mesenchymal progenitor cells (BMSC) are used for regenerating tissues of mesodermal origin, as well as tissues of different embryological derivation. Experimental evidence shows that BMSC are able to suppress the activation of the immune response by mechanisms that are still not completely understood. Thus far, in vitro studies carried using human or mouse cells indicate that autologous or allogeneic BMSC strongly suppress proliferation of T lymphocytes, triggered by cellular stimuli, nonspecific mitogenic stimuli, or antigenic peptides. Using cell proliferation and blocking assays, we demonstrated that BMSC inhibited the activation of murine splenocytes, T, and B lymphocytes. Direct contact of BMSC and target cells in a cognate fashion determined the inhibition of cell proliferation via engagement of the inhibitory molecule programmed death 1 (PD‐1) to its ligands PD‐L1 and PD‐L2, leading the target cells to modulate the expression of different cytokine receptors and transduction molecules for cytokine signaling. Soluble factors present on supernatants of BMSC cultures were effective in suppressing proliferation of B cells to a mitogenic stimulus. Taken together, these results highlight the complexity of the role of BMSC in regulating the immune response, asserting the possibility of their therapeutic application in transplantation and autoimmune diseases.

Development of sarcomas in mice implanted with mesenchymal stem cells seeded onto bioscaffolds

Bone marrow-derived mesenchymal stem cells (MSCs) are precursors of bone, cartilage and fat tissue. MSC can also regulate the immune response. For these properties, they are tested in clinical trials for tissue repair in combination with bioscaffolds or injected as cell suspension for immunosuppressant therapy. Experimental data, however, indicate that MSC can undergo or induce a tumorigenic process in determined circumstances. We used a modified model of ectopic bone formation in mice by subcutaneously implanting porous ceramic seeded with murine MSC. In this new model, host-derived sarcomas developed when we implanted MSC/bioscaffold constructs into syngeneic and immunodeficient recipients, but not in allogeneic hosts or when MSCs were injected as cell suspensions. The bioscaffold provided a tridimensional support for MSC to aggregate, thus producing the stimulus for triggering the process eventually leading to the transformation of surrounding cells and creating a surrogate tumor stroma. The chemical and physical characteristics of the bioscaffold did not affect tumor formation; sarcomas developed either when a stiff porous ceramic was used or when the scaffold was a smooth collagen sponge. The immunoregulatory function of MSC contributed to tumor development. Implanted MSC expanded clones of CD4+CD25+ T regulatory lymphocytes that suppressed hosts antitumor immune response.

Recruitment of a Host's Osteoprogenitor Cells Using Exogenous Mesenchymal Stem Cells Seeded on Porous Ceramic

The contribution of the hosts circulating progenitor cells after implantation of mesenchymal stem cells (MSC)/bioscaffold combinations for repairing bone defects has not been elucidated, although this issue affects the clinical application of the tissue engineering approach. We implanted blocks of hydroxyapatite loaded with murine MSCs into syngenic, allogenic, and immunocompromised recipients. After 8 weeks, we found that bone tissue was formed in syngenic and immunocompromised animals. The implanted cells appeared pivotal in the early stages of tissue development, but cells of the recipients origin finally made bone. In this system, osteoprogenitors migrated from the recipient to the implant, whereas the implanted cells left the scaffold and entered the circulatory flow. We observed rapid destruction of implanted cells when allogenic MSC/bioscaffold combinations were grafted onto immunocompetent recipients without immunosuppressant therapy. This destruction blocked the recruitment process and did not allow the formation of new bone tissue. The possibility that the implanted exogenous MSCs could engage the hosts osteoprogenitor cells to form new bone tissue could open new perspectives for the tissue engineering approach to bone repair, including the opportunity of using allogenic cells combined with a temporary immunosuppressant therapy, stimulating the replacement of the exogenous cells with autologous cells.

A humanized model of experimental autoimmune uveitis in HLA class II transgenic mice

Experimental autoimmune uveitis (EAU) is a disease of the neural retina induced by immunization with retinal antigens, such as interphotoreceptor retinoid-binding protein (IRBP) and arrestin (retinal soluble antigen, S-Ag). EAU serves as a model for human autoimmune uveitic diseases associated with major histocompatibility complex (HLA) genes, in which patients exhibit immunological responses to retinal antigens. Here we report the development of a humanized EAU model in HLA transgenic (TG) mice. HLA-DR3, -DR4, -DQ6, and -DQ8 TG mice were susceptible to IRBP-induced EAU. Importantly, HLA-DR3 TG mice developed severe EAU with S-Ag, to which wild-type mice are highly resistant. Lymphocyte proliferation was blocked by anti-HLA antibodies, confirming that antigen is functionally presented by the human MHC molecules. Disease could be transferred by immune cells with a Th1-like cytokine profile. Antigen-specific T cell repertoire, as manifested by responses to overlapping peptides derived from S-Ag or IRBP, differed from that of wild-type mice. Interestingly, DR3 TG mice, but not wild-type mice, recognized an immunodominant S-Ag epitope between residues 291 and 310 that overlaps with a region of S-Ag recognized by uveitis patients. Thus, EAU in HLA TG mice offers a new model of uveitis that should represent human disease more faithfully than currently existing models.

Mouse Models of Experimental Autoimmune Uveitis

The mouse model of experimental autoimmune uveitis, induced by immunization of mice with the retinal protein IRBP, was developed in our laboratory 20 years ago and published in 1988. Since that time it has been adopted by many investigators and has given rise to many studies that helped elucidate genetic influences, dissect the basic mechanisms of pathogenesis and test novel immunotherapeutic paradigms. The current overview will summarize the salient features of the experimental autoimmune uveitis model and discuss its mechanisms.

Mesenchymal Stem Cells Induce Functionally Active T-Regulatory Lymphocytes in a Paracrine Fashion and Ameliorate Experimental Autoimmune Uveitis

PURPOSE Mesenchymal stem/progenitor cells (MSCs) have regenerative and immunomodulatory properties, exerted by cell-cell contact and in a paracrine fashion. Part of their immunosuppressive activity has been ascribed to their ability to promote the induction of CD4+CD25+FoxP3+ T lymphocytes with regulatory functions (Treg). Here the authors studied the effect of MSCs on the induction of Treg and on the development of autoimmunity, and they examined the possibility that MSC-mediated Treg induction could be attributed to the secretion of soluble factors. METHODS The authors induced experimental autoimmune uveitis (EAU) in mice by immunization with the 1-20 peptide of the intraphotoreceptor binding protein. At the same time, some of the animals were treated intraperitoneally with syngeneic MSCs. The authors checked T-cell responses and in vitro Treg conversion by cell proliferation and blocking assays, in cell-cell contact and transwell settings. TGFβ and TGFβ receptor gene expression analyses were performed by real-time PCR. RESULTS The authors found that a single intraperitoneal injection of MSCs was able to significantly attenuate EAU and that a significantly higher percentage of adaptive Treg was present in MSC-treated mice than in MSC-untreated animals. In vitro blocking of antigen presentation by major histocompatibility complex class II precluded priming and clonal expansion of antigen-specific Treg, whereas blockade of TGFβ impaired the expression of FoxP3, preventing the conversion of CD4+ T cells into functionally active Treg. CONCLUSIONS The authors demonstrated that MSCs can inhibit EAU and that their immunomodulatory function is due at least in part to the induction of antigen-specific Treg in a paracrine fashion by secreting TGFβ.

When stem cells meet immunoregulation.

The clinical use of stem cells to prevent tissue injury or reconstruct damaged organs is constrained by different ethical and biological issues. Whereas the use of adult stem cells isolated from differentiated tissues is advantageous from the ethical point of view, the immune response of a host to implants of either embryonic or adult stem cells remains a critical problem. Embryonic stem cells can be rejected by an immunocompetent recipient as well as some types of adult stem cells. There is, however, a population of adult stem cells able to differentiate into the three mesenchymal lineages, osteocytes, chondrocytes, adipocytes that have the additional capacity of modulating the immune response by the activation of disparate mechanisms, among which the generation of antigen-specific CD4(+)CD25(+)FoxP3(+) regulatory T lymphocytes. This short review will focus on the immunological properties of embryonic and adult stem cells are, with particular emphasis on the immunomodulatory function of mesenchymal stem cells and their interactions with regulatory T lymphocytes.

Lipocalin-2 controls the expression of SDF-1 and the number of responsive cells in bone

Lipocalin-2 (LCN2) is a member of the lipocalin family, small secreted proteins functioning as modulators of many different physiological processes including cell differentiation, proliferation and apoptosis. LCN2 expression is also up-regulated in several pathological conditions, including inflammation and cancer. LCN2 synthesis has been described in epithelia, bone and cells of the immune system. Despite its wide expression the role of LCN2 remains to be fully elucidated. To better understand the role of this lipocalin in the bone/bone marrow system we generated transgenic mice over-expressing LCN2 specifically in bone under the control of a type I collagen promoter. In the bone marrow of these transgenic mice we observed an increased expression of SDF-1 that correlated with an increased number of CD34+/CXCR4+ (SDF-1 receptor) cells. To some extent, this appeared due to an enhanced cell proliferation rate. The higher level of the factor synthesis and the increased number of cells expressing its receptor was maintained during animal aging. Our results show that LCN2 could play a role in determining the number of CD34+/CXCR4+ precursor cells in the bone marrow thus contributing to the control of the bone marrow microenvironment.

Organization of Extracellular Matrix Fibers Within Polyglycolic Acid–Polylactic Acid Scaffolds Analyzed Using X-Ray Synchrotron-Radiation Phase-Contrast Micro Computed Tomography

Spatiotemporal organized patterns of cell surface-associated and extracellular matrix (ECM)-embedded molecules play important roles in the development and functioning of tissues. ECM proteins interact with the surface of bioscaffold polymers and influence material-driven control of cell differentiation., Using X-ray phase-contrast micro computed tomography (microCT), we visualized the three-dimensional (3D) image of ECM organization after in vitro seeding of bone marrow-derived human and murine mesenchymal stem cells (MSCs) induced to myogenic differentiation, labelled with iron oxide nanoparticles, and seeded onto polyglycolic acid-polylactic acid scaffolds. X-ray microCT enabled us to detect with high spatial resolution the 3D structural organization of ECM within the bioscaffold and how the presence of cells modified the construct arrangement. Species-specific differences between the matrix produced by human and murine cells were observed. In conclusion, X-ray synchrotron radiation microCT analysis appeared to be a useful tool to identify the spatiotemporal pattern of organization of ECM fibers within a bioscaffold.

Differentially Expressed Genes in MHC-Compatible Rat Strains That Are Susceptible or Resistant to Experimental Autoimmune Uveitis

PURPOSE Experimental autoimmune uveitis (EAU) is an established model for immune-mediated human uveitis. Although several genes from major histocompatibility complex (MHC) loci have been shown to play a role in uveitis, little is known about the role of non-MHC genes in the pathogenesis of EAU. Several non-MHC genes have been implicated in the pathogenesis of various autoimmune diseases. The primary objective of this study was to identify the non-MHC genes involved in the pathogenesis of EAU, to identify potential drug targets and possibly to target their protein products for immunotherapy. METHODS EAU was induced in the susceptible (Lewis; LEW) or resistant (Fischer 344; F344) rats that have identical MHC class II haplotype. Draining lymph node cells were obtained during the innate and adaptive phase of the immune response, and the pattern of gene expression was evaluated using microarray technology. Differentially expressed genes were validated at mRNA and protein levels using various methods. RESULTS Susceptibility to EAU was associated with an increased expression of numerous non-MHC genes such as Th1-type cytokines and chemokines, antiapoptotic factors, hormones, and neurotransmitters and a downregulation of selected adhesion molecules. In this study a combined genetic-genomic approach was used to identify different patterns of gene expression associated with the sensitization and effector phase of EAU pathogenesis. CONCLUSIONS The data demonstrate that the differential expression of several non-MHC genes is associated with the mechanism of uveitis.