The Peritoneum: Health, Disease, and Perspectives regarding Tissue Engineering and Cell Therapies.

There are several pathologies associated with the peritoneum, such as mesothelioma and peritonitis. Moreover, the peritoneum is widely used in ultrafiltration procedures, i.e., peritoneal dialysis, presenting advantages over hemodialysis. On the other hand, ultrafiltration failure may lead to dialysis-induced fibrosis and hypervolemia. Therefore, the pathophysiological study of this tissue is of extreme biomedical importance. Studies investigating the biology of the cells dwelling in the peritoneum wall provide evidence of their plasticity and progenitor features. For instance, both mesothelial and submesothelial cells present characteristics similar to mesenchymal stem cells, including osteogenic and adipogenic differentiation potential, support of extramedullary hematopoiesis, modulation of inflammatory responses, and regulation of tumor progression. Indeed, the participation of each cell type in peritoneal pathological and physiological phenomena is still under debate, especially regarding a possible differentiation pathway connecting these peritoneal cells. The primary aim of this review is to raise this discussion. In order to do so, we will firstly provide an overview of the peritoneum anatomy, histology, and ontology, and finally we will address how a better understanding of peritoneal cell biology may contribute to future cell therapy and tissue engineering approaches.

Lack of galectin-3 up-regulates IgA expression by peritoneal B1 lymphocytes during B cell differentiation.

Galectin-3 is a ß-galactoside-binding protein with an inhibitory role in B cell differentiation into plasma cells in distinct lymphoid tissues. We use a model of chronic schistosomiasis, a well-characterized experimental disease hallmarked by polyclonal B cell activation, in order to investigate the role of galectin-3 in controlling IgA production through peritoneal B1 cells. Chronically infected, galectin-3-deficient mice (Lgals3(-/-)) display peritoneal fluid hypercellularity, increased numbers of atypical peritoneal IgM(+)/IgA(+) B1a and B1b lymphocytes and histological disturbances in plasma cell niches when compared with Lgals3(+/+) mice. Similar to our infection model, peritoneal B1 cells from uninfected Lgals3(-/-) mice show enhanced switching to IgA after in vitro treatment with interleukin-5 plus transforming growth factor-ß (IL-5 + TGF-ß1). A higher number of IgA(+) B1a lymphocytes was found in the peritoneal cavity of Lgals3(-/-)-uninfected mice at 1 week after i.p. injection of IL-5 + TGF-ß1; this correlates with the increased levels of secreted IgA detected in the peritoneal fluid of these mice after cytokine treatment. Interestingly, a higher number of degranulated mast cells is present in the peritoneal cavity of uninfected and Schistosoma mansoni-infected Lgals3(-/-) mice, indicating that, at least in part, mast cells account for the enhanced differentiation of B1 into IgA-producing B cells found in the absence of galectin-3. Thus, a novel role is revealed for galectin-3 in controlling the expression of surface IgA by peritoneal B1 lymphocytes; this might have important implications for manipulating the mucosal immune response.

Platelet-rich plasma releasate differently stimulates cellular commitment toward the chondrogenic lineage according to concentration.

Platelet-rich plasma has been used to treat articular cartilage defects, with the expectations of anabolic and anti-inflammatory effects. However, its role on cellular chondrogenic or fibrogenic commitment is still a controversy. Herein, the role of platelet-rich plasma releasate, the product obtained following platelet-rich plasma activation, on cellular commitment toward the chondrogenic lineage was evaluated in vitro. Human nasoseptal chondrogenic cells and human bone marrow mesenchymal stromal cells were used as cell types already committed to the chondrogenic lineage and undifferentiated cells, respectively, as different concentrations of platelet-rich plasma releasate were tested in comparison to commonly used fetal bovine serum. Low concentration of platelet-rich plasma releasate (2.5%) presented similar effects on cellular growth compared to 10% fetal bovine serum, for both cell types. In a three-dimensional culture system, platelet-rich plasma releasate alone did not induce full nasoseptal chondrogenic cells cartilage-like pellet formation. Nonetheless, platelet-rich plasma releasate played a significant role on cell commitment as high-passage nasoseptal chondrogenic cells only originated cartilage-like pellets when expanded in the presence of platelet-rich plasma releasate rather than fetal bovine serum. Histological analyses and measurements of pellet area demonstrated that even low concentrations of platelet-rich plasma releasate were enough to prevent nasoseptal chondrogenic cells from losing their chondrogenic potential due to in vitro expansion thereby promoting their recommitment. Low concentration of platelet-rich plasma releasate supplemented in chondrogenic medium also increased the chondrogenic potential of mesenchymal stromal cells seeded on collagen-hyaluronic acid scaffolds, as observed by an increase in chondrogenic-related gene expression, sulfated glycosaminoglycan production, and compressive modulus following in vitro culture. On the contrary, higher concentration of platelet-rich plasma releasate (10%) hampered some of these features. In conclusion, platelet-rich plasma releasate was able to prevent cellular chondrogenic capacity loss, inducing regain of their phenotype, and modulate cell commitment. Our data support the hypothesis of platelet-rich plasma chondrogenic potential, allowing fetal bovine serum substitution for platelet-rich plasma releasate at specific concentrations in culture medium when chondrogenic commitment is desired on specific cell types and moments of culture.

The involvement of the spleen during chronic phase of Schistosoma mansoni infection in galectin-3-/- mice.

Schistosoma mansoni synthesizes glycoconjugates which interact with galectin-3, eliciting an intense humoral immune response. Moreover, it was demonstrated that galectin-3 regulates B cell differentiation into plasma cells. Splenomegaly is a hallmark event characterized by polyclonal B cell activation and enhancement of antibody production. Here, we investigated whether galectin-3 interferes with spleen organization and B cell compartment during chronic schistosomiasis, using wild type (WT) and galectin-3-/- mice. In chronically-infected galectin-3-/- mice the histological architecture of the spleen, including white and red pulps, was disturbed with heterogeneous lymphoid follicles, an increased number of plasma cells (CD19-B220-/lowCD138+) and a reduced number of macrophages (CD19-B220-Mac-1+CD138-) and B lymphocytes (CD19+B220+/highCD138-), compared with the WT infected mice. In the absence of galectin-3 there was an increase of annexin-V+PI- cells and a major presence of apoptotic cells in spleen compared with WT infected mice. In spleen of WT infected mice galectin-3 was largely expressed in lymphoid follicles and extrafollicular sites. Thus, we propose that galectin-3 plays a role in splenic architecture, controlling distinct events such as apoptosis, macrophage activity, B cell differentiation and plasmacytogenesis in the course of S. mansoni infection.

Lack of galectin-3 disturbs mesenteric lymph node homeostasis and B cell niches in the course of Schistosoma mansoni infection.

Galectin-3 is a ß-galactoside-binding protein that has been shown to regulate pathophysiological processes, including cellular activation, differentiation and apoptosis. Recently, we showed that galectin-3 acts as a potent inhibitor of B cell differentiation into plasma cells. Here, we have investigated whether galectin-3 interferes with the lymphoid organization of B cell compartments in mesenteric lymph nodes (MLNs) during chronic schistosomiasis, using WT and galectin-3(-/-) mice. Schistosoma mansoni synthesizes GalNAcß1-4(Fucα1-3)GlcNAc(Lac-DiNAc) structures (N-acetylgalactosamine ß1-4 N-acetylglucosamine), which are known to interact with galectin-3 and elicit an intense humoral response. Antigens derived from the eggs and adult worms are continuously drained to MLNs and induce a polyclonal B cell activation. In the present work, we observed that chronically-infected galectin-3(-/-) mice exhibited a significant reduced amount of macrophages and B lymphocytes followed by drastic histological changes in B lymphocyte and plasma cell niches in the MLNs. The lack of galectin-3 favored an increase in the lymphoid follicle number, but made follicular cells more susceptible to apoptotic stimuli. There were an excessive quantity of apoptotic bodies, higher number of annexin V(+)/PI(-) cells, and reduced clearance of follicular apoptotic cells in the course of schistosomiasis. Here, we observed that galectin-3 was expressed in non-lymphoid follicular cells and its absence was associated with severe damage to tissue architecture. Thus, we convey new information on the role of galectin-3 in regulation of histological events associated with B lymphocyte and plasma cell niches, apoptosis, phagocytosis and cell cycle properties in the MLNs of mice challenged with S.mansoni.

Interplay of cysteinyl leukotrienes and TGF-ß in the activation of hepatic stellate cells from Schistosoma mansoni granulomas.

Hepatic stellate cells (HSCs) have a critical role in liver physiology, and in the pathogenesis of liver inflammation and fibrosis. Here, we investigated the interplay between leukotrienes (LT) and TGF-ß in the activation mechanisms of HSCs from schistosomal granulomas (GR-HSCs). First, we demonstrated that GR-HSCs express 5-lipoxygenase (5-LO), as detected by immunolocalization in whole cells and confirmed in cell lysates through western blotting and by mRNA expression through RT-PCR. Moreover, mRNA expression of 5-LO activating protein (FLAP) and LTC(4)-synthase was also documented, indicating that GR-HSCs have the molecular machinery required for LT synthesis. Morphological analysis of osmium and Oil-Red O-stained HSC revealed large numbers of small lipid droplets (also known as lipid bodies). We observed co-localization of lipid droplet protein marker (ADRP) and 5-LO by immunofluorescence microscopy. We demonstrated that GR-HSCs were able to spontaneously release cysteinyl-LTs (CysLTs), but not LTB(4,) into culture supernatants. CysLT production was highly enhanced after TGF-ß-stimulation. Moreover, the 5-LO inhibitor zileuton and 5-LO gene deletion were able to inhibit the TGF-ß-stimulated proliferation of GR-HSCs, suggesting a role for LTs in HSC activation. Here, we extend the immunoregulatory function of HSC by demonstrating that HSC from liver granulomas of schistosome-infected mouse are able to release Cys-LTs in a TGF-ß-regulated manner, potentially impacting pathogenesis and liver fibrosis in schistosomiasis.

Galectin-3 regulates peritoneal B1-cell differentiation into plasma cells.

Extracellular galectin-3 participates in the control of B2 lymphocyte migration and adhesion and of their differentiation into plasma cells. Here, we analyzed the role of galectin-3 in B1-cell physiology and the balance between B1a and B1b lymphocytes in the peritoneal cavity. In galectin-3(-/-) mice, the total number of B1a lymphocytes was lower, while B1b lymphocyte number was higher as compared to wild-type mice. The differentiation of B1a cells into plasma cells was associated with their abnormal adhesion and location on the mesentery. The B220 and CD43, constitutively expressed by B1 lymphocytes, were respectively up- and downregulated in galectin-3(-/-) mice. Mononuclear cells were strongly adhered to the mesenteric membranes of both CD43(-/-) and galectin-3(-/-) mice, but in contrast to CD43(-/-) mice, the accumulation of B1 cells in peritoneal membranes in galectin-3(-/-) mice was accompanied by their functional differentiation into plasma cells. We have shown that in the absence of galectin-3, B1-cell differentiation into plasma cells is favored and the dynamic equilibrium of B1-cell populations in the peritoneum is maintained through a compensatory increase in B1b lymphocytes.

Bone marrow subendosteal microenvironment harbours functionally distinct haemosupportive stromal cell populations.

In adult animals, bone marrow is the major site of blood cell production, which is controlled by interactions between the local stroma and blood cell progenitors. The endosteal/subendosteal environment comprises bone-lining and adjacent reticular cells and sustains haemopoietic stem cell (HSC) self-renewal, proliferation and differentiation. We have questioned the specific role of each of these stroma cells in controlling HSC fate. We have isolated two distinct stroma-cell populations containing subendosteal reticulocytes (F-RET) and osteoblasts (F-OST) from periosteum-free fragments of murine femurs by a two-step collagenase-digestion procedure. Both populations produce similar extracellular matrix (collagen I, laminin, fibronectin, decorin), except for collagen IV, which is low in F-OST. They also express osteogenic markers: osteopontin, osteonectin, bone sialoprotein and alkaline phosphatase (ALP). The quantity and activity of ALP are however higher in F-OST. When co-cultured with bone marrow mononuclear cells or lineage-negative haemopoietic progenitors, F-OST stroma induces low proliferation and high maintenance of early haemopoietic progenitors, whereas F-RET stroma induces high short-term proliferation and differentiation. Analysis by reverse transcription/polymerase chain reaction has revealed higher levels of Jagged-1 expression by F-OST cells than by the F-RET population. Thus, two adjacent stroma cells (subendosteal and endosteal) play distinct roles in controlling the stem-cell capacity and fate of HSC and probably contribute distinctly to HSC niche formation.

Haemopoietic progenitors in the adult mouse omentum: permanent production of B lymphocytes and monocytes.

The coelome-associated lympho-myeloid tissues, including the omentum, are derived from early embryo haemopoietic tissue of the splanchnopleura, and produce B lymphocytes and macrophages. They are reactive in pathologies involving coelomic cavities, in which they can expand in situ the cells of inflammatory infiltrates. We have addressed the question of the role of the adult omentum in permanent basal production of early lymphopoietic progenitors (pro-B/pre-B cells), through characterisation of omentum cells ex vivo, and study of their in vitro differentiation. We have shown that the murine omentum produces early haemopoietic progenitors throughout life, including B-cell progenitors prior to the Ig gene recombination expressing RAG-1 and lambda5, as well as macrophages. Their production is stroma-dependent. The omentum stroma can supply in vitro the cytokines (SDF-1alpha, Flt3 ligand and IL-7) and the molecular environment required for generation of these two cell lineages. Omentum haemopoietic progenitors are similar to those observed in foetal blood cell production, rather than to progenitors found in the adult haemopoietic tissue in the bone marrow--in terms of phenotype expression and differentiation capacity. We conclude that a primitive pattern of haemopoiesis observed in the early embryo is permanently preserved and functional in the adult omentum, providing production of cells engaged in nonspecific protection of abdominal intestinal tissue and of the coelomic cavity.

Connexin expression and gap-junction-mediated cell interactions in an in vitro model of haemopoietic stroma.

In addition to the steady-state production of all blood cells, bone marrow can respond to an increased requirement for one or several cell lineages. The hormonal controls involved may act directly on blood cell progenitors or indirectly through modification of the haemopoietic environment. Intercellular gap junctions formed by connexins (Cx) provide direct communication among adjacent cells and the functional integration of multicellular systems. Since haemopoietic stroma is determinant for blood cell production, we have questioned whether gap-junction-dependent controls of haemopoiesis are sensitive to hormones and vitamins. We have analysed the expression, synthesis, cell distribution and formation of functional gap junctions in the murine bone-marrow stroma cell line S-17, and between stromal cells and blood cell progenitors. Nine Cxs were identified by reverse transcription/polymerase chain reaction, and only Cx43 by Western blot and immunofluorescence. All of the studied parameters were sensitive to intrinsic controls dependent upon the pattern of cell growth and modulated by exogenous controls mediated by retinol and steroids. Positive or negative modulation was specific for different Cxs. FACS analysis showed communication among the stromal cells and between stromal cells and myeloid (Mac1+) but not lymphoid (B220+) progenitors. Calcein transfer modulation did not correspond to the modulation of Cx43 expression and formation of connexons, suggesting the participation of other Cxs. Thus, functional gap junctions among haemopoietic stroma cells and between stroma and haematopoietic cells in the bone marrow may be modulated in response to hormonal stimuli, potentially controlling overall blood cell production.

Myelopoiesis in the omentum of normal mice and during abdominal inflammatory processes.

Coelomic cavities are relatively isolated from the systemic circulation of blood cells. Resident cell populations have a proper phenotype and kinetics, maintaining their steady-state populations and their responsiveness to local inflammatory reactions, in which the number and quality of coelomic cells can be greatly increased and modified. We have addressed the question of whether the increase in cell infiltrate in the inflamed abdominal cavity is sustained by the proliferation of myeloid cells in the omentum, and if so what are the characteristics of the progenitor cells involved and how the omentum controls their proliferation and differentiation. In the omentum under normal conditions and with inflammation due to schistosomal infection we found that pluripotent early myeloid progenitors were capable of giving rise to all the myeloid lineages in clonogenic assays, but not to the totipotent blood stem cells. Besides the major haemopoietins (GM-CSF, M-CSF, G-CSF, IL-5), the omentum stroma constitutively expressed SDF-1 alpha, the chemokine which elicits homing of circulating early haemopoietic progenitors. While normal omentum stroma produced LIF, its expression was substituted by SCF in inflamed tissues. In the first situation a slow steady-state renewal of progenitors is potentially favoured, while their intense expansion may be predominant in the latter one. We propose that the increase in cells in the abdominal cavity in inflammatory reactions is due to the enhanced input and expansion of early myeloid progenitors sustaining the in situ production of abdominal cell populations, rather than to the input of systemic circulating inflammatory cells.