Bone formation by human paediatric marrow stromal cells in a functional allogeneic immune system.

Allogeneic stem-cell based regenerative medicine is a promising approach for bone defect repair. The use of chondrogenically differentiated human marrow stromal cells (MSCs) has been shown to lead to bone formation by endochondral ossification in immunodeficient pre-clinical models. However, an insight into the interactions between the allogeneic immune system and the human MSC-derived bone grafts has not been fully achieved yet. The choice of a potent source of MSCs isolated from pediatric donors with consistent differentiation and high proliferation abilities, as well as low immunogenicity, could increase the chance of success for bone allografts. In this study, we employed an immunodeficient animal model humanised with allogeneic immune cells to study the immune responses towards chondrogenically differentiated human pediatric MSCs (ch-pMSCs). We show that ch-differentiated pMSCs remained non-immunogenic to allogeneic CD4 and CD8 T cells in an in vitro co-culture model. After subcutaneous implantation in mice, ch-pMSC-derived grafts were able to initiate bone mineralisation in the presence of an allogeneic immune system for 3 weeks without the onset of immune responses. Re-exposing the splenocytes of the humanised animals to pMSCs did not trigger further T cell proliferation, suggesting an absence of secondary immune responses. Moreover, ch-pMSCs generated mature bone after 8 weeks of implantation that persisted for up to 6 more weeks in the presence of an allogeneic immune system. These data collectively show that human allogeneic chondrogenically differentiated pediatric MSCs might be a safe and potent option for bone defect repair in the tissue engineering and regenerative medicine setting.

The response of human macrophages to 3D printed titanium antibacterial implants does not affect the osteogenic differentiation of hMSCs.

Macrophage responses following the implantation of orthopaedic implants are essential for successful implant integration in the body, partly through intimate crosstalk with human marrow stromal cells (hMSCs) in the process of new bone formation. Additive manufacturing (AM) and plasma electrolytic oxidation (PEO) in the presence of silver nanoparticles (AgNPs) are promising techniques to achieve multifunctional titanium implants. Their osteoimmunomodulatory properties are, however, not yet fully investigated. Here, we studied the effects of implants with AgNPs on human macrophages and the crosstalk between hMSCs and human macrophages when co-cultured in vitro with biofunctionalised AM Ti6Al4V implants. A concentration of 0.3 g/L AgNPs in the PEO electrolyte was found to be optimal for both macrophage viability and inhibition of bacteria growth. These specimens also caused a decrease of the macrophage tissue repair related factor C-C Motif Chemokine Ligand 18 (CCL18). Nevertheless, co-cultured hMSCs could osteogenically differentiate without any adverse effects caused by the presence of macrophages that were previously exposed to the PEO (±AgNPs) surfaces. Further evaluation of these promising implants in a bony in vivo environment with and without infection is highly recommended to prove their potential for clinical use.

In Vitro Mineralisation of Tissue-Engineered Cartilage Reduces Endothelial Cell Migration, Proliferation and Tube Formation.

Tissue engineering bone via endochondral ossification requires the generation of a cartilage template which undergoes vascularisation and remodelling. While this is a promising route for bone repair, achieving effective cartilage vascularisation remains a challenge. Here, we investigated how mineralisation of tissue-engineered cartilage affects its pro-angiogenic potential. To generate in vitro mineralised cartilage, human mesenchymal stromal cell (hMSC)-derived chondrogenic pellets were treated with ß-glycerophosphate (BGP). After optimising this approach, we characterised the changes in matrix components and pro-angiogenic factors by gene expression analysis, histology and ELISA. Human umbilical vein endothelial cells (HUVECs) were exposed to pellet-derived conditioned media, and migration, proliferation and tube formation were assessed. We established a reliable strategy to induce in vitro cartilage mineralisation, whereby hMSC pellets are chondrogenically primed with TGF-ß for 2 weeks and BGP is added from week 2 of culture. Cartilage mineralisation determines loss of glycosaminoglycans, reduced expression but not protein abundance of collagen II and X, and decreased VEGFA production. Finally, the conditioned medium from mineralised pellets showed a reduced ability to stimulate endothelial cell migration, proliferation and tube formation. The pro-angiogenic potential of transient cartilage is thus stage-dependent, and this aspect must be carefully considered in the design of bone tissue engineering strategies.

Chondrogenically Primed Human Mesenchymal Stem Cells Persist and Undergo Early Stages of Endochondral Ossification in an Immunocompetent Xenogeneic Model.

Tissue engineering approaches using progenitor cells such as mesenchymal stromal cells (MSCs) represent a promising strategy to regenerate bone. Previous work has demonstrated the potential of chondrogenically primed human MSCs to recapitulate the process of endochondral ossification and form mature bone in vivo, using immunodeficient xenogeneic models. To further the translation of such MSC-based approaches, additional investigation is required to understand the impact of interactions between human MSC constructs and host immune cells upon the success of MSC-mediated bone formation. Although human MSCs are considered hypoimmunogenic, the potential of chondrogenically primed human MSCs to induce immunogenic responses in vivo, as well as the efficacy of MSC-mediated ectopic bone formation in the presence of fully competent immune system, requires further elucidation. Therefore, the aim of this study was to investigate the capacity of chondrogenically primed human MSC constructs to persist and undergo the process of endochondral ossification in an immune competent xenogeneic model. Chondrogenically differentiated human MSC pellets were subcutaneously implanted to wild-type BALB/c mice and retrieved at 2 and 12 weeks post-implantation. The percentages of CD4+ and CD8+ T cells, B cells, and classical/non-classical monocyte subsets were not altered in the peripheral blood of mice that received chondrogenic MSC constructs compared to sham-operated controls at 2 weeks post-surgery. However, MSC-implanted mice had significantly higher levels of serum total IgG compared to sham-operated mice at this timepoint. Flow cytometric analysis of retrieved MSC constructs identified the presence of T cells and macrophages at 2 and 12 weeks post-implantation, with low levels of immune cell infiltration to implanted MSC constructs detected by CD45 and CD3 immunohistochemical staining. Despite the presence of immune cells in the tissue, MSC constructs persisted in vivo and were not degraded/resorbed. Furthermore, constructs became mineralised, with longitudinal micro-computed tomography imaging revealing an increase in mineralised tissue volume from 4 weeks post-implantation until the experimental endpoint at 12 weeks. These findings indicate that chondrogenically differentiated human MSC pellets can persist and undergo early stages of endochondral ossification following subcutaneous implantation in an immunocompetent xenogeneic model. This scaffold-free model may be further extrapolated to provide mechanistic insight to osteoimmunological processes regulating bone regeneration and homeostasis.

Bio-inspired polymeric iron-doped hydroxyapatite microspheres as a tunable carrier of rhBMP-2.

Hybrid superparamagnetic microspheres with bone-like composition, previously developed by a bio-inspired assembling/mineralization process, are evaluated for their ability to uptake and deliver recombinant human bone morphogenetic protein-2 (rhBMP-2) in therapeutically-relevant doses along with prolonged release profiles. The comparison with hybrid non-magnetic and with non-mineralized microspheres highlights the role of nanocrystalline, nanosize mineral phases when they exhibit surface charged groups enabling the chemical linking with the growth factor and thus moderating the release kinetics. All the microspheres show excellent osteogenic ability with human mesenchymal stem cells whereas the hybrid mineralized ones show a slow and sustained release of rhBMP-2 along 14 days of soaking into cell culture medium with substantially bioactive effect, as reported by assay with C2C12 BRE-Luc cell line. It is also shown that the release extent can be modulated by the application of pulsed electromagnetic field, thus showing the potential of remote controlling the bioactivity of the new micro-devices which is promising for future application of hybrid biomimetic microspheres in precisely designed and personalized therapies.

Cartilage Oligomeric Matrix Protein-Derived Peptides Secreted by Cartilage Do Not Induce Responses Commonly Observed during Osteoarthritis.

OBJECTIVE: To evaluate if 3 peptides derived from the cartilage oligomeric matrix protein (COMP), which wounded zones of cartilage secrete into synovial fluid, possess biological activity and might therefore be involved in the regulation of specific aspects of joint regeneration. METHODS: The 3 peptides were produced by chemical synthesis and then tested in vitro for known functions of the COMP C-terminal domain from which they derive, and which are involved in osteoarthritis: transforming growth factor-ß (TGF-ß) signaling, vascular homeostasis, and inflammation. Results. None of the peptides affected the gene expression of COMP in osteochondral progenitor cells (P > 0.05). We observed no effects on the vascularization potential of endothelial cells (P > 0.05). In cultured synovium explants, no differences on the expression of catabolic enzymes or proinflammatory cytokines were found when peptides were added (P > 0.05). DISCUSSION AND CONCLUSIONS: The 3 peptides tested do not regulate TGF-ß signaling, angiogenesis and vascular tube formation, or synovial inflammation in vitro and therefore most likely do not play a major role in the disease process.

Allogeneic Chondrogenic Mesenchymal Stromal Cells Alter Helper T Cell Subsets in CD4+ Memory T Cells.

Implantation of chondrogenically differentiated mesenchymal stromal cells (MSCs) leads to bone formation in vivo through the process of endochondral ossification. The use of allogeneic MSCs for this purpose may be a promising new approach to replace the current gold standard of bone regeneration. However, the success of using allogeneic cells depends on the interaction between the implanted cells and the host's endogenous immune cells. Th17 T cells and other CD4 helper T cell subtypes have been shown to negatively impact chondrogenesis, however, it is unclear how the interaction between these cells affects bone regeneration mediated by these cells. The aim of the current work was to assess the effect of chondrogenic MSC pellets on Th1, Th2, Th17, and regulatory T cells in vitro. Human MSCs were nonchondrogenic (-TGFß3) and chondrogenically (+TGFß3) differentiated for 7 or 21 days. Memory T cells (sorted from the CD4 population of peripheral blood mononuclear cells [PBMCs]), as well as total PBMCs were cocultured with allogeneic nonchondrogenic and chondrogenic MSC pellets for 3 days. Seven-day differentiated allogeneic nonchondrogenic and chondrogenic MSC pellets that were cocultured with memory T cells resulted in a significant increase in Th2 and a decrease in Th1 T cells. Furthermore, the co-culture of 21-day differentiated nonchondrogenic and chondrogenic MSC pellets with memory T cells resulted in a significant increase in Th2 and Th17 T cells, as well as a decrease in Th1 and regulatory T cells. Interleukin (IL)-6 was identified as a predominant cytokine involved in this interaction between allogeneic chondrogenically differentiated MSC pellets and memory CD4 T cells, with high levels of IL-6 being secreted in the supernatants of this cocultured condition. The findings of this study highlight the potential of chondrogenically differentiated MSC pellets to alter the ratio of Th1 and Th2 as well as Th17 and regulatory T cell subsets. Additional analysis investigating bone formation by chondrogenically differentiated MSCs in an allogeneic setting may identify a novel role of these T cell subsets in bone regeneration processes mediated by chondrogenically differentiated MSCs. Impact statement Allogeneic mesenchymal stromal cells (MSCs) have the potential to be an off-the-shelf treatment for bone repair. However, the lack of knowledge of the immune cells involved in this process has hampered the progression to the clinic. The current study has shown that allogeneic chondrogenic MSCs have the potential to skew the ratio of specific helper CD4 T cell subsets in vitro. This has now provided insight for future in vivo experiments to investigate the role of these T cell subsets in the early stages of bone regeneration mediated by allogeneic chondrogenic MSCs.

Pediatric Mesenchymal Stem Cells Exhibit Immunomodulatory Properties Toward Allogeneic T and B Cells Under Inflammatory Conditions.

Mesenchymal stem cells from pediatric patients (pMSCs) are an attractive cell source in regenerative medicine, due to their higher proliferation rates and better differentiation abilities compared to adult MSCs (aMSCs). We have previously characterized the immunomodulatory abilities of pMSCs on T cells under co-culture. It has also been reported that aMSCs can inhibit B cell proliferation and maturation under inflammatory conditions. In this study, we therefore aimed to clarify the immunomodulatory effect of pMSCs toward T and B cells in an inflammatory microenvironment. Bone marrow derived pMSCs were primed to simulate inflammatory conditions by exposure with 50 ng/mL of IFN-γ for 3 days. To analyze the interaction between pMSCs and T cells, CD3/CD28 stimulated peripheral blood mononuclear cells (PBMCs) were co-cultured with primed or unprimed pMSCs. To investigate B cell responses, quiescent B cells obtained from spleens by CD43 negative selection were stimulated with anti-IgM, anti-CD40, IL-2, and co-cultured with either IFN-γ primed or unprimed pMSC. pMSC phenotype, B and T cell proliferation, and B cell functionality were analyzed. Gene expression of indoleamine 2,3-dioxygenease (IDO), as well as the expression of HLA-ABC, HLA-DR and the co-stimulatory molecules CD80 and CD86 was upregulated on pMSCs upon IFN-γ priming. IFN-γ did not alter the immunomodulatory abilities of pMSCs upon CD4+ nor CD8+ stimulated T cells compared to unprimed pMSCs. IFN-γ primed pMSCs but not unprimed pMSCs strongly inhibited naïve (CD19+CD27-), memory (CD19+CD27+), and total B cell proliferation. Antibody-producing plasmablast (CD19+CD27highCD38high) formation and IgG production were also significantly inhibited by IFN-γ primed pMSCs compared to unprimed pMSCs. Collectively, these results show that pMSCs have immunomodulatory effects upon the adaptive immune response which can be potentiated by inflammatory stimuli. This knowledge is useful in regenerative medicine and allogeneic transplantation applications toward tailoring pMSCs function to best modulate the immune response for a successful implant engraftment and avoidance of a strong immune reaction.

Follistatin Effects in Migration, Vascularization, and Osteogenesis in vitro and Bone Repair in vivo.

The use of biomaterials and signaling molecules to induce bone formation is a promising approach in the field of bone tissue engineering. Follistatin (FST) is a glycoprotein able to bind irreversibly to activin A, a protein that has been reported to inhibit bone formation. We investigated the effect of FST in critical processes for bone repair, such as cell recruitment, osteogenesis and vascularization, and ultimately its use for bone tissue engineering. In vitro, FST promoted mesenchymal stem cell (MSC) and endothelial cell (EC) migration as well as essential steps in the formation and expansion of the vasculature such as EC tube-formation and sprouting. FST did not enhance osteogenic differentiation of MSCs, but increased committed osteoblast mineralization. In vivo, FST was loaded in an in situ gelling formulation made by alginate and recombinant collagen-based peptide microspheres and implanted in a rat calvarial defect model. Two FST variants (FST288 and FST315) with major differences in their affinity to cell-surface proteoglycans, which may influence their effect upon in vivo bone repair, were tested. In vitro, most of the loaded FST315 was released over 4 weeks, contrary to FST288, which was mostly retained in the biomaterial. However, none of the FST variants improved in vivo bone healing compared to control. These results demonstrate that FST enhances crucial processes needed for bone repair. Further studies need to investigate the optimal FST carrier for bone regeneration.

Novel In Situ Gelling Hydrogels Loaded with Recombinant Collagen Peptide Microspheres as a Slow-Release System Induce Ectopic Bone Formation.

New solutions for large bone defect repair are needed. Here, in situ gelling slow release systems for bone induction are assessed. Collagen-I based Recombinant Peptide (RCP) microspheres (MSs) are produced and used as a carrier for bone morphogenetic protein 2 (BMP-2). The RCP-MSs are dispersed in three hydrogels: high mannuronate (SLM) alginate, high guluronate (SLG) alginate, and thermoresponsive hyaluronan derivative (HApN). HApN+RCP-MS forms a gel structure at 32 ºC or above, while SLM+RCP-MS and SLG+RCP-MS respond to shear stress displaying thixotropic behavior. Alginate formulations show sustained release of BMP-2, while there is minimal release from HApN. These formulations are injected subcutaneously in rats. SLM+RCP-MS and SLG+RCP-MS loaded with BMP-2 induce ectopic bone formation as revealed by X-ray tomography and histology, whereas HApN+RCP-MS do not. Vascularization occurs within all the formulations studied and is significantly higher in SLG+MS and HApN+RCP-MS than in SLM+RCP-MS. Inflammation (based on macrophage subset staining) decreases over time in both alginate groups, but increases in the HApN+RCP-MS condition. It is shown that a balance between inflammatory cell infiltration, BMP-2 release, and vascularization, achieved in the SLG+RCP-MS alginate condition, is optimal for the induction of de novo bone formation.

Isolating Pediatric Mesenchymal Stem Cells with Enhanced Expansion and Differentiation Capabilities.

Mesenchymal stem cells/marrow stromal cells (MSCs) are attractive for applications ranging from research and development to use in clinical therapeutics. However, the most commonly studied MSCs, adult bone marrow MSCs (A-MSCs), are limited by significant donor variation resulting in inconsistent expansion rates and multilineage differentiation capabilities. We have recently obtained permission to isolate pediatric MSCs (P-MSCs) from surplus iliac crest bone chips. Here, we developed a simple and easily replicable isolation protocol yielding P-MSCs, which adhere to MSC defining guidelines. After confirming immunophenotypic marker expression, we compared expansion rates, senescence, morphology, and trilineage differentiation of P-MSCs to A-MSCs for multiple donors. We found P-MSCs have faster in vitro replication, consistently show significantly lower senescence, and are capable of more reproducible multilineage differentiation than A-MSCs. We, therefore, believe P-MSCs are a promising candidate for use in research applications and potentially as part of an allogeneic therapeutic treatment.

Data on the surface morphology of additively manufactured Ti-6Al-4V implants during processing by plasma electrolytic oxidation.

Additively manufactured Ti-6Al-4V implants were biofunctionalized using plasma electrolytic oxidation. At various time points during this process scanning electron microscopy imaging was performed to analyze the surface morphology (van Hengel et al., 2017) [1]. This data shows the changes in surface morphology during plasma electrolytic oxidation. Data presented in this article are related to the research article "Selective laser melting porous metallic implants with immobilized silver nanoparticles kill and prevent biofilm formation by methicillin-resistant Staphylococcus aureus" (van Hengel et al., 2017) [1].

Selective laser melting porous metallic implants with immobilized silver nanoparticles kill and prevent biofilm formation by methicillin-resistant Staphylococcus aureus.

Implant-associated infection and limited longevity are two major challenges that orthopedic devices need to simultaneously address. Additively manufactured porous implants have recently shown tremendous promise in improving bone regeneration and osseointegration, but, as any conventional implant, are threatened by infection. In this study, we therefore used rational design and additive manufacturing in the form of selective laser melting (SLM) to fabricate porous titanium implants with interconnected pores, resulting in a 3.75 times larger surface area than corresponding solid implants. The SLM implants were biofunctionalized by embedding silver nanoparticles in an oxide surface layer grown using plasma electrolytic oxidation (PEO) in Ca/P-based electrolytes. The PEO layer of the SLM implants released silver ions for at least 28 days. X-ray diffraction analysis detected hydroxyapatite on the SLM PEO implants but not on the corresponding solid implants. In vitro and ex vivo assays showed strong antimicrobial activity of these novel SLM PEO silver-releasing implants, without any signs of cytotoxicity. The rationally designed SLM porous implants outperformed solid implants with similar dimensions undergoing the same biofunctionalization treatment. This included four times larger amount of released silver ions, two times larger zone of inhibition, and one additional order of magnitude of reduction in numbers of CFU in an ex vivo mouse infection model.

Enamel Matrix Derivative has No Effect on the Chondrogenic Differentiation of Mesenchymal Stem Cells.

BACKGROUND: Treatment of large bone defects due to trauma, tumor resection, or congenital abnormalities is challenging. Bone tissue engineering using mesenchymal stem cells (MSCs) represents a promising treatment option. However, the quantity and quality of engineered bone tissue are not sufficient to fill large bone defects. The aim of this study was to determine if the addition of enamel matrix derivative (EMD) improves in vitro chondrogenic priming of MSCs to ultimately improve in vivo MSC mediated endochondral bone formation. METHODS: MSCs were chondrogenically differentiated in 2.0 × 10(5) cell pellets in medium supplemented with TGFß3 in the absence or presence of 1, 10, or 100 µg/mL EMD. Samples were analyzed for gene expression of RUNX2, Col II, Col X, and Sox9. Protein and glycoaminoglycan (GAG) production were also investigated via DMB assays, histology, and immunohistochemistry. Osteogenic and adipogenic differentiation capacity were also assessed. RESULTS: The addition of EMD did not negatively affect chondrogenic differentiation of adult human MSCs. EMD did not appear to alter GAG production or expression of chondrogenic genes. Osteogenic and adipogenic differentiation were also unaffected though a trend toward decreased adipogenic gene expression was observed. CONCLUSION: EMD does not affect chondrogenic differentiation of adult human MSCs. As such the use of EMD in combination with chondrogenically primed MSCs for periodontal bone tissue repair is unlikely to have negative effects on MSC differentiation.

Endoplasmic reticulum stress, unfolded protein response and altered T cell differentiation in necrotizing enterocolitis.

BACKGROUND: Endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) play important roles in chronic intestinal inflammation. Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in preterm infants and is characterized by acute intestinal inflammation and necrosis. The objective of the study is to investigate the role of ER stress and the UPR in NEC patients. METHODS: Ileal tissues from NEC and control patients were obtained during surgical resection and/or at stoma closure. Splicing of XBP1 was detected using PCR, and gene expression was quantified using qPCR and Western blot. RESULTS: Splicing of XBP1 was only detected in a subset of acute NEC (A-NEC) patients, and not in NEC patients who had undergone reanastomosis (R-NEC). The other ER stress and the UPR pathways, PERK and ATF6, were not activated in NEC patients. A-NEC patients showing XBP1 splicing (A-NEC-XBP1s) had increased mucosal expression of GRP78, CHOP, IL6 and IL8. Similar results were obtained by inducing ER stress and the UPR in vitro. A-NEC-XBP1s patients showed altered T cell differentiation indicated by decreased mucosal expression of RORC, IL17A and FOXP3. A-NEC-XBP1s patients additionally showed more severe morphological damage and a worse surgical outcome. Compared with A-NEC patients, R-NEC patients showed lower mucosal IL6 and IL8 expression and higher mucosal FOXP3 expression. CONCLUSIONS: XBP1 splicing, ER stress and the UPR in NEC are associated with increased IL6 and IL8 expression levels, altered T cell differentiation and severe epithelial injury.

High beta-palmitate fat controls the intestinal inflammatory response and limits intestinal damage in mucin Muc2 deficient mice.

BACKGROUND: Palmitic-acid esterified to the sn-1,3 positions of the glycerol backbone (alpha, alpha'-palmitate), the predominant palmitate conformation in regular infant formula fat, is poorly absorbed and might cause abdominal discomfort. In contrast, palmitic-acid esterified to the sn-2 position (beta-palmitate), the main palmitate conformation in human milk fat, is well absorbed. The aim of the present study was to examine the influence of high alpha, alpha'-palmitate fat (HAPF) diet and high beta-palmitate fat (HBPF) diet on colitis development in Muc2 deficient (Muc2(-/-)) mice, a well-described animal model for spontaneous enterocolitis due to the lack of a protective mucus layer. METHODS: Muc2(-/-) mice received AIN-93G reference diet, HAPF diet or HBPF diet for 5 weeks after weaning. Clinical symptoms, intestinal morphology and inflammation in the distal colon were analyzed. RESULTS: Both HBPF diet and AIN-93G diet limited the extent of intestinal erosions and morphological damage in Muc2(-/-) mice compared with HAPF diet. In addition, the immunosuppressive regulatory T (Treg) cell response as demonstrated by the up-regulation of Foxp3, Tgfb1 and Ebi3 gene expression levels was enhanced by HBPF diet compared with AIN-93G and HAPF diets. HBPF diet also increased the gene expression of Pparg and enzymatic antioxidants (Sod1, Sod3 and Gpx1), genes all reported to be involved in promoting an immunosuppressive Treg cell response and to protect against colitis. CONCLUSIONS: This study shows for the first time that HBPF diet limits the intestinal mucosal damage and controls the inflammatory response in Muc2(-/-) mice by inducing an immunosuppressive Treg cell response.

Mucin Muc2 deficiency and weaning influences the expression of the innate defense genes Reg3ß, Reg3γ and angiogenin-4.

BACKGROUND: Mucin Muc2 is the structural component of the intestinal mucus layer. Absence of Muc2 leads to loss of this layer allowing direct bacterial-epithelial interactions. We hypothesized that absence of the mucus layer leads to increased expression of innate defense peptides. Specifically, we aimed to study the consequence of Muc2 deficiency (Muc2(-/-)) on the expression of regenerating islet-derived protein 3 beta (Reg3ß), regenerating islet-derived protein 3 gamma (Reg3γ), and angiogenin-4 (Ang4) in the intestine shortly before and after weaning. METHODS: Intestinal tissues of Muc2(-/-) and wild-type (WT) mice were collected at postnatal day 14 (P14, i.e. pre-weaning) and P28 (i.e. post-weaning). Reg3ß, Reg3γ, and Ang4 expression was studied by quantitative real-time PCR, Western-blot, in situ hybridization, and immunohistochemistry. RESULTS: Reg3ß and Reg3γ were expressed by diverging epithelial cell types; namely enterocytes, Paneth cells, and goblet cells. Additionally, Ang4 expression was confined to Paneth cells and goblet cells. Expression of Reg3ß, Reg3γ, and Ang4 differed between WT and Muc2(-/-) mice before and after weaning. Interestingly, absence of Muc2 strongly increased Reg3ß and Reg3γ expression in the small intestine and colon. Finally, morphological signs of colitis were only observed in the distal colon of Muc2(-/-) mice at P28, where and when expression levels of Reg3ß, Reg3γ, and Ang4 were the lowest. CONCLUSIONS: Expression of Reg3 proteins and Ang4 by goblet cells point to an important role for goblet cells in innate defense. Absence of Muc2 results in up-regulation of Reg3ß and Reg3γ expression, suggesting altered bacterial-epithelial signaling and an innate defense response in Muc2(-/-) mice. The inverse correlation between colitis development and Reg3ß, Reg3γ, and Ang4 expression levels might point toward a role for these innate defense peptides in regulating intestinal inflammation.

GATA-4/-6 and HNF-1/-4 families of transcription factors control the transcriptional regulation of the murine Muc5ac mucin during stomach development and in epithelial cancer cells.

During human embryonic and fetal development of the gastrointestinal tract, the gene encoding the MUC5AC mucin has a spatio-temporal pattern of expression restricted to the stomach. In order to better understand the molecular mechanisms responsible for this restricted pattern of expression, we have studied Muc5ac expression in the developing stomach of the mouse and correlated it to that of transcription factors known to be involved in cell differentiation programs during development. Our results indicate that GATA-6 and HNF-4α expression increased concomitantly with the induction of Muc5ac expression in embryonic stomach. We then studied Muc5ac transcriptional regulation by these transcription factors and showed that they all transactivate Muc5ac promoter. We also identified several active GATA-4/-5/-6 and HNF-1/-4 cis-elements using gel shift assays, chromatin immunoprecipitation and site-directed mutagenesis. Among all Muc5ac regulators, only GATA-6 and HNF-4a expression was concomitant to that of Muc5ac in the developing stomach. This is thus in favor of an important role for these two transcription factors as regulators of expression of the Muc5ac mucin during stomach development and in epithelial cancer cells.

Colonic gene expression patterns of mucin Muc2 knockout mice reveal various phases in colitis development.

BACKGROUND: Mucin Muc2 knockout (Muc2(-/-)) mice spontaneously develop colitis. METHODS: To identify genes and biological responses which play a pivotal role during colitis development in Muc2(-/-) mice, gene expression profiles of colonic tissues from 2- and 4-week-old Muc2(-/-) and wildtype mice were determined using microarrays. RESULTS: The majority of highly upregulated genes in 2-week-old as well as 4-week-old Muc2(-/-) mice were primarily involved in immune responses related to antigen processing/presentation, B-cell and T-cell receptor signaling, leukocyte transendothelial migration, and Jak-STAT signaling. Specifically, Muc2(-/-) mice expressed high levels of immunoglobulins, murine histocompatibility-2, proinflammatory cytokines, chemokines, and antimicrobial proteins. Additionally, in 4-week-old Muc2(-/-) mice, expression of genes involved in cell structure related pathways was significantly altered. Particularly, the tight junction-associated gene claudin-10 was upregulated, whereas claudin-1 and claudin-5 were downregulated. Furthermore, 4-week-old Muc2(-/-) mice showed increased expression of genes regulating cell growth in conjunction with increased crypt length and increased epithelial proliferation. CONCLUSIONS: Muc2-deficiency leads to an active inflammatory response in 2- and 4-week-old Muc2(-/-) mice as demonstrated by the altered expression in immune response related genes. In addition, 4-week-old Muc2(-/-) mice also showed a decrease in epithelial barrier function and an increase in epithelial proliferation as indicated by, respectively, the altered expression in tight junction-related genes and upregulation of genes stimulating cell growth. Remarkably, upregulation of genes stimulating cell growth correlated with increased crypt length and increased epithelial proliferation in 4-week-old Muc2(-/-) mice. Together, these data demonstrate that there are distinct phases in colitis development in 2-4-week-old Muc2(-/-) mice.