Mesenchymal stem cells tune the development of monocyte-derived dendritic cells toward a myeloid-derived suppressive phenotype through growth-regulated oncogene chemokines.

Mesenchymal stem/stromal cells (MSCs) are promising potential candidates for the treatment of immunological diseases because of their immunosuppressive functions. However, the molecular mechanisms that mediate MSCs' immunosuppressive activity remain elusive. In this article, we report for the first time, to our knowledge, that secreted growth-regulated oncogene (GRO) chemokines, specifically GRO-γ, in human MSC-conditioned media have an effect on the differentiation and the function of human monocyte-derived dendritic cells. The monocyte-derived dendritic cells were driven toward a myeloid-derived suppressor cell (MDSC)-like phenotype by the GRO chemokines. GRO-γ-treated MDSCs had a tolerogenic phenotype that was characterized by an increase in the secretion of IL-10 and IL-4, and a reduction in the production of IL-12 and IFN-γ. We have also shown that the mRNA expression levels of the arginase-1 and inducible NO synthase genes, which characterize MDSCs, were upregulated by GRO-γ-primed mouse bone marrow cells. In addition, the ability of GRO-γ-treated bone marrow-derived dendritic cells to stimulate the OVA-specific CD8(+) T (OT-1) cell proliferation and the cytokine production of IFN-γ and TNF-α were significantly decreased in vivo. Our findings allow a greater understanding of how MDSCs can be generated and offer new perspectives to exploit the potential of MDSCs for alternative approaches to treat chronic inflammation and autoimmunity, as well as for the prevention of transplant rejection.

Mesenchymal stem cells promote neutrophil activation by inducing IL-17 production in CD4+ CD45RO+ T cells.

Mesenchymal stem cells (MSCs) are multi-potent with numerous mesenchymal-lineage differentiation potential and immunomodulatory capabilities. However, the immunoregulatory properties of MSCs are not clearly defined. The objective of the present study was to elucidate the role(s) of MSCs in IL-17 production and the subsequent effect(s) on neutrophil activation. We have demonstrated that human bone marrow-derived MSCs (BM-MSCs) instruct anti-CD3/anti-CD28 antibody-activated CD4(+) CD45RO(+) memory T cells, but not other CD4(+) subsets or CD8(+) T cells, to produce IL-17 after cell-cell contact. After the addition of IL-17, neutrophil phagocytic activity was increased. This is the first report on the ability of BM-MSCs to induce IL-17 production in memory CD4(+) T cells that, in turn, promotes enhanced phagocytic activity of neutrophils. These results suggest that MSCs regulate the functional activation of neutrophils via their role in modulating IL-17 from CD4(+) CD45RO(+) memory T cells.

Cytokine profiles in various graft-versus-host disease target organs following hematopoietic stem cell transplantation.

Previous studies using genetic-deficient murine models suggest that different T-helper subsets may contribute to different types of tissue damages in graft-versus-host disease (GvHD). However, there is limited information available on the distribution of T-helper cytokines in the various GvHD target tissues. In the current study, an acute GvHD murine model was set up to directly assess the in situ cytokine profiles in various GvHD tissue lesions; in addition, we also studied GvHD tissues from patients who had undergone bone marrow transplantation procedures. We observed that interferon-γ (IFN-γ was dominant in murine liver and gastrointestinal tissue lesions, whereas IFN-γ and interleukin 17 (IL-17) were abundant in murine skin lesions. Furthermore, in human GvHD tissues, interleukin 4 (IL-4) and IFN-γ were predominant in liver lesions and colon lesions, respectively, while no specific cytokine was prevalent in human GvHD skin lesions. In addition, a low ratio of CD4(+) T helper (Th) versus CD8(+) T cytotoxic (Tc) cells in human GvHD tissue lesions, especially in the liver, was detected, and this contrasts with the situation in murine GvHD tissues where CD4(+) Th cells were predominant. Dual staining for CD markers and cytokine expression showed that IFN-γ-secreting T cells were enriched in all murine GvHD target tissue lesions, and Tc1 and Tc2 cells were predominant in human GvHD colon and liver sections, respectively. However, IFN-γ(+) Th1, IL-17(+) Th17, IFN-γ(+) Tc1, and IL-17(+) Tc17 cells were slightly more frequent in human skin lesions compared to IL-4(+) Th2 and IL-4(+) Tc2 cells. To sum up, these results suggest that differences in cytokine imbalances may significantly contribute to tissue-specific pathogenesis in GvHD target organs, and CD8(+) Tc cells may play an important role in human GvHD induction.

Matrix stiffness regulation of integrin-mediated mechanotransduction during osteogenic differentiation of human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) cultured on extracellular matrices with different stiffness have been shown to possess diverse lineage commitment owing to the extracellular mechanical stimuli sensed by the cells. The aim of this study was to further delineate how matrix stiffness affects intracellular signaling through the mechanotransducers Rho kinase (ROCK) and focal adhesion kinase (FAK) and subsequently regulates the osteogenic phenotype of MSCs. MSCs were cultured in osteogenic medium on tunable polyacrylamide hydrogels coated with type I collagen with elasticities corresponding to Young's modulus of 7.0 ± 1.2 and 42.1 ± 3.2 kPa. Osteogenic differentiation was increased on stiffer matrices, as evident by type I collagen, osteocalcin, and Runx2 gene expressions and alizarin red S staining for mineralization. Western blot analysis demonstrated an increase in kinase activities of ROCK, FAK, and ERK1/2 on stiffer matrices. Inhibition of FAK, an important mediator of osteogenic differentiation, and inhibition of ROCK, a known mechanotransducer of matrix stiffness during osteogenesis, resulted in decreased expression of osteogenic markers during osteogenic induction. In addition, FAK affects osteogenic differentiation through ERK1/2, whereas ROCK regulates both FAK and ERK1/2. Furthermore, α(2)-integrin was upregulated on stiffer matrices during osteogenic induction, and its knockdown by siRNA downregulated the osteogenic phenotype through ROCK, FAK, and ERK1/2. Taken together, our results provide evidence that the matrix rigidity affects the osteogenic outcome of MSCs through mechanotransduction events that are mediated by α(2)-integrin.

Mesenchymal stem cells negatively regulate dendritic lineage commitment of umbilical-cord-blood-derived hematopoietic stem cells: an unappreciated mechanism as immunomodulators.

Due to their immunomodulatory functions, mesenchymal stem cells (MSCs) have great potential for clinical applications to prevent rejection in organ transplantation and to prevent graft-versus-host disease in hematopoietic stem cell (HSC) transplantation. Since dendritic cells (DCs) play an important role in modulating diverse T cell responses, including rejection and graft-versus-host disease, the goal of this study was to investigate whether MSCs modulate DC differentiation from HSCs and if this effect could be one of the mechanisms for MSCs' immune-modulating functions. Our results demonstrate that differentiation of HSCs into mature DCs is inhibited in the presence of MSCs. Similar frequency of dendritic precursors in the cultures, either with or without MSCs, suggests that the inhibition of MSCs on the differentiation of mature DCs from HSCs could be due to the arresting of maturation at the dendritic precursor step. Reduced levels of cyclic AMP, adenosine 3',5'-cyclic monophosphate (cAMP) and beta-catenin in DC-like cells from the cocultures are detected, suggesting that induction of apoptosis and inhibition of differentiation could be the basis for the inhibition of mature DCs from HSCs by MSCs. Further, our results demonstrate that DCs derived from HSCs in the presence of MSCs are functionally impaired, especially for those after direct contact with MSCs. To investigate the basis of functional impairment, our data show downregulated tumor necrosis factor-alpha and transforming growth factor-beta1 secretion and upregulated interleukin-6 (IL6) and IL1beta secretion in the cultures with MSCs. Together, MSCs can inhibit differentiation of mature DCs from HSCs by arresting them at the precursor stage and induce their apoptosis. Further, HSC-derived DCs in the presence of MSCs are functionally impaired, which could be partly due to the upregulation of IL6 secretion.

Characterization of enolase allergen from Rhodotorula mucilaginosa.

Rhodotorula mucilaginosa (also known as R. rubra) is among the most commonly found yeast strains in our environment. However, allergens from R. mucilaginosa have not yet been characterized at the molecular level. The purpose of this study was to characterize the enolase allergen from R. mucilaginosa and examine the allergenic/antigenic cross-reactivity among fungal enolases. The full-length cDNA encoding the R. mucilaginosa enolase was isolated through the reverse transcriptase-polymerase chain reaction in conjunction with the 5'-end and 3'-end rapid amplification cDNA end reactions. The corresponding natural enolase from R. mucilaginosa was identified using two-dimensional gel electrophoresis and N-terminal amino acid sequence analysis. The results showed that the enolase from R. mucilaginosa is a protein of 439 residues and is encoded by a cDNA of 1497 bp. It shares high sequence identity with enolase allergens from Candida albicans (85%), Saccharomyces cerevisiae (76%), Penicillium citrinum (76%), Aspergillus fumigatus (76%), Cladosporium herbarum (76.5%), and Alternaria alternata (74%). A 47-kD component in the R. mucilaginosa extracts was found to react with IgE or rabbit anti-enolase antiserum and has an N-terminal amino acid sequence identical to that deduced from the isolated enolase cDNA. Sera from three (21%) of 14 allergic patients sensitized to R. mucilaginosa showed IgE binding to this 47-kD R. mucilaginosa component and the His-tagged recombinant enolase. A rabbit antiserum against the P. citrinum enolase and a monoclonal antibody (MoAb; Afueno 8) against the A. fumigatus enolase reacted with all 5 fungal enolases tested. However, an MoAb (E2a) generated by using the Saccharomyces enolase as antigen could only recognize the immunizing enolase. In addition, heterogeneity in immunoblot profiles of IgE antibodies in serum samples from 9 allergic patients against 5 different fungal enolases tested was also observed. The presence of IgE cross-reactivity among enolase allergens from R. mucilaginosa, C. albicans and P. citrinum was detected by immunoblot inhibition. In conclusion, a new and cross-reactive enolase allergen from R. mucilaginosa (Rho m 1) was identified. Although enolases are highly conserved allergens among different fungal species, most of the allergic patients examined in this study differed in their IgE reactivity to the 5 different fungal enolases tested. The results obtained will be of value in understanding the role of enolase allergen in clinical mould allergy.

cDNA cloning and immunological characterization of a newly identified enolase allergen from Penicillium citrinum and Aspergillus fumigatus.

BACKGROUND: Penicillium citrinum and Aspergillus fumigatus are prevalent indoor airborne fungal species that have been implicated in human respiratory allergic disorders. It is important to understand the allergenic profile of these fungal species. The purpose of the present study is to characterize a newly identified enolase allergen from P. citrinum and A. fumigatus. METHODS: Fungal proteins were separated by two-dimensional (2D) gel electrophoresis and blotted onto polyvinylidene difluoride membranes. Protein spots that reacted with IgE antibodies in serum samples from asthmatic patients were identified and the N-terminal amino acid sequences were determined by Edman degradation. The peptide sequences obtained were utilized in cloning the cDNA of the allergen genes by reverse transcriptase-polymerase chain reaction and the 5'- and 3'-rapid amplification cDNA end reactions. RESULTS: Our results from 2D immunoblotting identified a 47-kD IgE-reactive component in the extracts of P. citrinum and A. fumigatus. The N-terminal amino acid sequences of the 47-kD proteins are homologous to those of fungal enolases. The corresponding enolase cDNA from P. citrinum contains 1,552 bp and encodes a protein of 438 residues. In A. fumigatus, the isolated enolase cDNA has 1,649 bp and contains a 438-amino acid open reading frame. The deduced amino acid sequences of these two enolases have 94% identity. These enolases from P. citrinum and A. fumigatus were expressed in Escherichia coli as a His-tagged protein and designated as rPen c 22 and rAsp f 22, respectively. Sera from 7 (30%) of the 23 Penicillium-sensitized asthmatic patients showed IgE binding to the 47-kD P. citrinum component (Pen c 22) and rPen c 22. In addition, six of seven Pen c 22-positive serum samples have IgE immunoblot reactivity to the 47-kD A. fumigatus component (Asp f 22) and rAsp f 22. A polyclonal rabbit antiserum generated against the N-terminal peptide of Pen c 22 can react with Pen c 22, rPen c 22, Asp f 22 and rAsp f 22. In addition, the presence of IgE cross-reactivity between rPen c 22 and rAsp f 22 and between enolases from A. fumigatus and Alternaria alternata was also detected by immunoblot inhibition. CONCLUSIONS: These results demonstrated that a novel enolase allergen from P. citrinum (Pen c 22) and A. fumigatus (Asp f 22) was identified. In addition, IgE cross-reactivity between enolase allergens from A. fumigatus and P. citrinum and between enolases from A. fumigatus and A. alternata was also detected. Results obtained provide more information on fungal enolase allergens.