Mesenchymal Stem Cell-Derived Extracellular Vesicles as Therapeutics and as a Drug Delivery Platform.

Mesenchymal stem cells (MSCs) are one of the most easily accessible stem cells that can be obtained from various human tissues. They have raised considerable interests for their potential applications in tissue repair, anti-cancer therapy, and inflammation suppression. Stem cell-based therapy was first used to treat muscular dystrophies and has been studied intensively for its efficacy in various disease models, including myocardial infarction, kidney injuries, liver injuries, and cancers. In this review, we summarized the potential mechanisms underlying MSC-derived EVs therapy as a drug delivery platform. Additionally, based on currently published data, we predicted a potential therapeutic role of cargo proteins shuttled by EVs from MSCs. These data may support the therapeutic strategy of using the MSC-derived EVs to accelerate this strategy from bench to bedside. Stem Cells Translational Medicine 2019;8:880&886.

Taurodeoxycholate Increases the Number of Myeloid-Derived Suppressor Cells That Ameliorate Sepsis in Mice.

Bile acids (BAs) control metabolism and inflammation by interacting with several receptors. Here, we report that intravenous infusion of taurodeoxycholate (TDCA) decreases serum pro-inflammatory cytokines, normalizes hypotension, protects against renal injury, and prolongs mouse survival during sepsis. TDCA increases the number of granulocytic myeloid-derived suppressor cells (MDSCLT) distinctive from MDSCs obtained without TDCA treatment (MDSCL) in the spleen of septic mice. FACS-sorted MDSCLT cells suppress T-cell proliferation and confer protection against sepsis when adoptively transferred better than MDSCL. Proteogenomic analysis indicated that TDCA controls chromatin silencing, alternative splicing, and translation of the immune proteome of MDSCLT, which increases the expression of anti-inflammatory molecules such as oncostatin, lactoferrin and CD244. TDCA also decreases the expression of pro-inflammatory molecules such as neutrophil elastase. These findings suggest that TDCA globally edits the proteome to increase the number of MDSCLT cells and affect their immune-regulatory functions to resolve systemic inflammation during sepsis.

Hepatitis C Virus-Induced Myeloid-Derived Suppressor Cells Suppress NK Cell IFN-γ Production by Altering Cellular Metabolism via Arginase-1.

The hepatitis C virus (HCV) infects ∼ 200 million people worldwide. The majority of infected individuals develop persistent infection, resulting in chronic inflammation and liver disease, including cirrhosis and hepatocellular carcinoma. The ability of HCV to establish persistent infection is partly due to its ability to evade the immune response through multiple mechanisms, including suppression of NK cells. NK cells control HCV replication during the early phase of infection and regulate the progression to chronic disease. In particular, IFN-γ produced by NK cells limits viral replication in hepatocytes and is important for the initiation of adaptive immune responses. However, NK cell function is significantly impaired in chronic HCV patients. The cellular and molecular mechanisms responsible for impaired NK cell function in HCV infection are not well defined. In this study, we analyzed the interaction of human NK cells with CD33(+) PBMCs that were exposed to HCV. We found that NK cells cocultured with HCV-conditioned CD33(+) PBMCs produced lower amounts of IFN-γ, with no effect on granzyme B production or cell viability. Importantly, this suppression of NK cell-derived IFN-γ production was mediated by CD33(+)CD11b(lo)HLA-DR(lo) myeloid-derived suppressor cells (MDSCs) via an arginase-1-dependent inhibition of mammalian target of rapamycin activation. Suppression of IFN-γ production was reversed by l-arginine supplementation, consistent with increased MDSC arginase-1 activity. These novel results identify the induction of MDSCs in HCV infection as a potent immune evasion strategy that suppresses antiviral NK cell responses, further indicating that blockade of MDSCs may be a potential therapeutic approach to ameliorate chronic viral infections in the liver.

Phytoncide Extracted from Pinecone Decreases LPS-Induced Inflammatory Responses in Bovine Mammary Epithelial Cells.

Mastitis is a prevalent inflammatory disease that remains one of the main causes of poor quality of milk. Phytoncides are naturally occurring anti-inflammatory compounds derived from plants and trees. To determine if treatment with phytoncide could decrease the severity of lipopolysaccharide (LPS)-induced inflammatory responses, mammary alveolar epithelial cells (MAC-T) were pretreated with phytoncide (0.02% and 0.04% (v/v)) followed by LPS treatment (1 and 25 µg/ml). The results demonstrated that phytoncide downregulated LPS-induced pro-inflammatory cyclooxygenase-2 (COX-2) expression. Additionally, LPS-induced activation of ERK1/2, p38, and Akt was attenuated by phytoncide. Treatment of cells with known pharmacological inhibitors of ERK1/2 (PD98059), p38 (SB203580), and Akt (LY294002) confirmed the association of these signaling pathways with the observed alterations in COX-2 expression. Moreover, phytoncide attenuated LPS-induced NF-κB activation and superoxide production, and, finally, treatment with phytoncide increased Nrf2 activation. Results suggest that phytoncide can decrease LPS-induced inflammation in MAC-T cells.

Shogaol overcomes TRAIL resistance in colon cancer cells via inhibiting of survivin.

In this study, we showed the ability of representative shogaol, which as a major component of ginger, to overcome TRAIL resistance by increasing apoptosis in colon cancer cells. Shogaol increased death receptor 5 (DR5) levels. Furthermore, shogaol decreased the expression level of antiapoptotic proteins (survivin and Bcl-2) and increased pro-apoptotic protein, Bax. Shogaol treatment induced apoptosis and a robust reduction in the levels of the antiapoptotic protein survivin but did not affect the levels of many other apoptosis regulators. Moreover, knockdown of survivin sensitized colon cancer cells to resistant of TRAIL-induced apoptosis. Therefore, we showed the functions of shogaol as a sensitizing agent to induce cell death of TRAIL-resistant colon cancer cells. This study gives rise to the possibility of applying shogaol as an antitumor agent that can be used for the purpose of combination treatment with TRAIL in TRAIL-resistant colon tumor therapy.

Phenethyl isothiocyanate sensitizes glioma cells to TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) is a promising antitumor therapy. However, many cancer cells, including malignant glioma cells, tend to be resistant to TRAIL, highlighting the need for strategies to overcome TRAIL resistance. Here we show that in combination with phenethyl isothiocyanate (PEITC), exposure to TRAIL induced apoptosis in TRAIL-resistant glioma cells. Subtoxic concentrations of PEITC significantly potentiated TRAIL-induced cytotoxicity and apoptosis in glioma cells. PEITC dramatically upregulated DR5 receptor expression but had no effects on DR4 receptor. PEITC enhances TRAIL-induced apoptosis through the downregulation of cell survival proteins and the upregulation of DR5 receptors through actions on the ROS-induced-p53.

Transcriptional regulation of IFN-λ genes in hepatitis C virus-infected hepatocytes via IRF-3·IRF-7·NF-κB complex.

Hepatitis C virus (HCV) infection in hepatocytes stimulates innate antiviral responses including the production of type III interferons (IFN-λ), including IL-28A, IL-28B, and IL-29. However, the molecular mechanism(s) regulating the expression of IFN-λ genes in HCV-infected hepatocytes remains undefined. In this study, we examined regulatory elements involved in the induction of IFN-λ genes following HCV infection in hepatocytes and further determined the binding of specific transcription factor(s) to promoter regions of IFN-λ genes. Our studies reveal that the regulatory portion for IL-28A, IL-28B, and IL-29 genes is localized to a 1-kb region in their respective promoters. Notably, interferon regulatory factor (IRF)-3 and -7 are the key transcriptional factors for the induction of IL-28A and IL-28B genes, whereas NF-κB is an additional requirement for the induction of the IL-29 gene. Ligation of Toll-like receptors (TLR) 3, 7, 8, and 9, which also activate IRFs and NF-κB, resulted in more robust production of IFN-λ than that observed with HCV infection, verifying the importance of TLR pathways in IFN-λ production. Furthermore, the addition of IFN-λ to HCV-infected hepatocytes decreased viral replication and produced a concurrent reduction in microRNA-122 (miR-122). The decrease in viral replication was enhanced by the co-administration of IFN-λ and miR-122 inhibitor (miRIDIAN), suggesting that such combinatorial therapies may be beneficial for the treatment of chronic HCV infection.

Hepatitis C virus promotes T-helper (Th)17 responses through thymic stromal lymphopoietin production by infected hepatocytes.

UNLABELLED: Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. Here we report that infection of hepatic cells by HCV stimulates nuclear factor kappa B (NFκB)-dependent production of thymic stromal lymphopoietin (TSLP). Hepatocyte-derived TSLP in turn conditions dendritic cells (DCs) to drive T-helper (Th)17 differentiation. The TSLP secreted by HCV-infected hepatoma cells is capable of activating human monocyte-derived DCs by up-regulating the expression of CD40, CD86, CCL17, CCL22, and CCL20 which are activating markers of DCs. In addition, the production of key cytokines for Th17 differentiation, transforming growth factor beta (TGF-ß), interleukin (IL)-6, and IL-21, is enhanced by human monocytes upon coculture with HCV-infected cells. Importantly, the blockade of TSLP using neutralizing antibody prevented the activation and maturation of DCs as well as the production of Th17 differentiation cytokines. DC conditioning by TSLP secreted from HCV-infected cells activated naïve CD4+ T lymphocytes, resulting in Th17 differentiation. Furthermore, we can detect substantial levels of hepatocyte TSLP in fibrotic liver tissue from chronic HCV patients. Thus, blockade of TSLP released by HCV-infected hepatocytes may suppress the induction/maintenance of hepatic Th17 responses and halt the progression of chronic liver disease to fibrosis and liver failure. CONCLUSION: Hepatocyte-derived TSLP conditions DCs to drive Th17 differentiation. Treatment of TSLP neutralizing antibody in HCV-infected hepatocyte/DC coculture abrogates DC conditioning and thereby inhibits Th17 differentiation.

Thymic stromal lymphopoietin is induced by respiratory syncytial virus-infected airway epithelial cells and promotes a type 2 response to infection.

BACKGROUND: Respiratory viral infection, including respiratory syncytial virus (RSV) and rhinovirus, has been linked to respiratory disease in pediatric patients, including severe acute bronchiolitis and asthma exacerbation. OBJECTIVE: The study examined the role of the epithelial-derived cytokine thymic stromal lymphopoietin (TSLP) in the response to RSV infection. METHODS: Infection of human airway epithelial cells was used to examine TSLP induction after RSV infection. Air-liquid interface cultures from healthy children and children with asthma were also tested for TSLP production after infection. Finally, a mouse model was used to directly test the role of TSLP signaling in the response to RSV infection. RESULTS: Infection of airway epithelial cells with RSV led to the production of TSLP via activation of an innate signaling pathway that involved retinoic acid induced gene I, interferon promoter-stimulating factor 1, and nuclear factor-κB. Consistent with this observation, airway epithelial cells from asthmatic children a produced significantly greater levels of TSLP after RSV infection than cells from healthy children. In mouse models, RSV-induced TSLP expression was found to be critical for the development of immunopathology. CONCLUSION: These findings suggest that RSV can use an innate antiviral signaling pathway to drive a potentially nonproductive immune response and has important implications for the role of TSLP in viral immune responses in general.

Myeloid suppressor cells induced by hepatitis C virus suppress T-cell responses through the production of reactive oxygen species.

UNLABELLED: Impaired T-cell responses in chronic hepatitis C virus (HCV) patients have been reported to be associated with the establishment of HCV persistent infection. However, the mechanism for HCV-mediated T-cell dysfunction is yet to be defined. Myeloid-derived suppressor cells (MDSCs) play a pivotal role in suppressing T-cell responses. In this study we examined the accumulation of MDSCs in human peripheral blood mononuclear cells (PBMCs) following HCV infection. We found that CD33(+) mononuclear cells cocultured with HCV-infected hepatocytes, or with HCV core protein, suppress autologous T-cell responses. HCV core-treated CD33(+) cells exhibit a CD14(+) CD11b(+/low) HLADR(-/low) phenotype with up-regulated expression of p47(phox) , a component of the NOX2 complex critical for reactive oxygen species (ROS) production. In contrast, immunosuppressive factors, arginase-1 and inducible nitric oxide synthase (iNOS), were not up-regulated. Importantly, treatment with an inactivator of ROS reversed the T-cell suppressive function of HCV-induced MDSCs. Lastly, PBMCs of chronic HCV patients mirror CD33(+) cells following treatment with HCV core where CD33(+) cells are CD14(+) CD11b(+) HLADR(-/low) , and up-regulate the expression of p47(phox). CONCLUSION: These results suggest that HCV promotes the accumulation of CD33(+) MDSC, resulting in ROS-mediated suppression of T-cell responsiveness. Thus, the accumulation of MDSCs during HCV infection may facilitate and maintain HCV persistent infection.

TSLP enhances the function of helper type 2 cells.

The cytokine thymic stromal lymphopoietin (TSLP) has been implicated in the development and progression of allergic inflammation in both humans and mice. TSLP has been shown to promote a Th2-type response through upregulation of OX40L on dendritic cells, and through direct induction of IL-4 production in naïve CD4+ T cells. However, its direct effect on effector Th cells has not been extensively investigated. In this study, we show that the level of TSLP receptor (TSLPR) expression on mouse effector Th2 cells is higher than on Th1 and Th17 cells, and that TSLP induced proliferation of effector Th2, but not Th1 nor Th17 cells. TSLP also induced the phosphorylation of signal transducer and activator of transcription (Stat) 5, and expression of the anti-apoptotic factor Bcl-2 in Th2 cells. Finally, TSLP-mediated proliferation on Th2 cells was enhanced by TCR stimulation, through IL-4-mediated induction of TSLPR expression. Taken together, these results indicate that TSLP is involved in exacerbation of mouse Th2-mediated allergic inflammation in a Th2 environment through direct stimulation of Th2 effector cells.

Accessibility control of TCR Vγ region by STAT5.

The signal of the IL-7R and signal transducers and activators of transcription (STAT) 5 plays an essential role in gammadelta T-cell development by inducing V-J recombination in the TCRgamma locus. Previously, we have shown that STAT5 binds to the Jgamma promoters and controls chromatin accessibility by histone acetylation. However, little is known on control mechanism of Vgamma region by the IL-7R. To elucidate the regulation by STAT5, we first analyzed the chromatin status of Vgamma region in primary thymocytes. The levels of histone H3 acetylation are high at Vgamma5, HsA element and Vgamma2 in Rag2(-/-) thymocytes but low in IL-7R alpha-chain (IL-7Ralpha)-deficient early thymocytes, suggesting that IL-7R signaling controls the accessibility of the Vgamma region. In addition, high levels of histone H3 acetylation and germ line transcription were induced at Vgamma5 and HsA by cytokine and STAT5 in cytokine-dependent Ba/F3 and other hematopoietic cell lines. Importantly, the chromatin accessibility of Vgamma5 gene is increased by cytokine signal. Furthermore, STAT5 was not recruited to a non-canonical STAT-binding motif in the endogenous chromatin of the Vgamma5 promoter by cytokine stimulation, while STAT5 binds to a consensus motif in the HsA element. In accordance with this result, STAT5 does not directly activate the Vgamma5 promoter by reporter assay. These results suggested that while STAT5 directly binds to HsA element and induces its histone acetylation, STAT5 indirectly activates the Vgamma5 promoter. Thus, this study implies a potential role of STAT5 in accessibility control of Vgamma region, especially at Vgamma5 and HsA.

TSLP production by epithelial cells exposed to immunodeficiency virus triggers DC-mediated mucosal infection of CD4+ T cells.

Mucosal dendritic cells have been implicated in the capture, storage, and transmission of HIV to CD4(+) T cells as well as in the promotion of HIV replication in activated CD4(+) T cells during the cognate T-cell and DC interaction. We report that HIV induces human genital mucosal epithelial cells to produce thymic stromal lymphopoietin (TSLP) via activation of the NFkappaB signaling pathway. The TSLP secreted by HIV exposed epithelial cells activated DC, which promoted proliferation and HIV-1 replication of co-cultured autologous CD4(+) T cells. In rhesus macaques, we observed dramatic increases in TSLP expression concurrent with an increase in viral replication in the vaginal tissues within the first 2 weeks after vaginal SIV exposure. These data suggest that HIV-mediated TSLP production by mucosal epithelial cells is a critical trigger for DC-mediated amplification of HIV-infection in activated CD4(+) T cells. The cross talk between mucosal epithelial cells and DC, mediated by HIV-induced TSLP, may be an important mechanism for the high rate of HIV infection in women through the vaginal mucosa.

Cutting edge: Inhibition of NF-kappaB-mediated TSLP expression by retinoid X receptor.

The epithelial-derived cytokine thymic stromal lymphopoietin (TSLP) has important roles in the initiation of allergic airway inflammation and the activation of dendritic cells. We have shown that the human TSLP gene is regulated in a NF-kappaB-dependent manner; however the factors that negatively regulate TSLP expression are not known. In this study we demonstrate that 9-cis-retinoic acid (9-cis-RA) is a negative regulator of TSLP expression in airway epithelial cells. This inhibition is manifested as a block in the IL-1beta-mediated recruitment of NF-kappaB to the human TSLP promoter. 9-cis-RA-mediated inhibition is not restricted to TSLP gene expression but rather reflects a general inhibition of NF-kappaB activation, as other NF-kappaB-regulated-genes were also inhibited in a similar manner by 9-cis-RA treatment. Taken as a whole, these data demonstrate that inhibition of IL-1beta-dependent genes by active retinoid X receptors involves antagonism of NF-kappaB signaling.

Induction of the IL-7 receptor alpha chain in mouse peripheral B cells by glucocorticoids.

Expression of the IL-7R alpha chain (IL-7R alpha) is strictly regulated during development and maturation of lymphocytes. While T cells express the IL-7R alpha in the periphery, B cells do not. Glucocorticoids (GCs) have pleiotypic effects on development and function of lymphocytes. Although GCs induce the transcription of IL-7R alpha gene in T cells, their effect on B cells is largely unknown. Here, we show that GCs induce the transcription and expression of IL-7R alpha in mouse peripheral B cells. This effect does not require de novo protein synthesis, because a protein synthesis inhibitor, cycloheximide, does not block the transcription. IL-7R signal pathway is intact in peripheral B cells because Stat5, one of the signal molecules of the IL-7R alpha, is phosphorylated by IL-7 stimulation. We also observed that IL-7 simulation induces the transcription of Cis-1, one of the target genes of Stat5. Furthermore, GC-induced IL-7R alpha can transmit survival signal in B cells. Therefore, this study demonstrates that GCs induce the transcription and expression of functional IL-7R alpha in peripheral B cells, and implies a potential role of the IL-7R in survival of B cells.

Inducible expression of the proallergic cytokine thymic stromal lymphopoietin in airway epithelial cells is controlled by NFkappaB.

The epithelial-derived cytokine thymic stromal lymphopoietin (TSLP) is important for the initiation of allergic airway inflammation through a dendritic cell-mediated T helper 2 response. To identify the factors that control TSLP expression, we examined the ability of inflammatory mediators to regulate TSLP production in human airway epithelial cells. We found that both IL-1beta and TNF-alpha were capable of inducing rapid TSLP production in primary human bronchial airway epithelial cells. We further characterized the human TSLP gene promoter, using two human epithelial cell lines, 16HBEo(-) and A549, and showed that IL-1beta- and TNF-alpha-mediated human TSLP promoter activation in these cells was mediated by an upstream NFkappaB site. Mutation of this NFkappaB site abolished activation, as did overexpression of a dominant-negative version of IkappaB kinase (IKK)beta (a kinase acting on IkappaB, the inhibitor of NFkappaB). Interestingly, human TSLP mRNA levels were also increased after exposure to Toll-like receptor (TLR) 2, TLR8, and TLR9 ligands, further supporting an important role for NFkappaB in TSLP gene regulation. Similarly, analysis of the mouse TSLP gene promoter revealed the presence of a similarly situated NFkappaB site that was also critical for IL-1beta-inducible expression of mouse TSLP. Taken together, these results demonstrate that the inflammatory mediators IL-1beta and TNF-alpha regulate human TSLP gene expression in an NFkappaB-dependent manner.

Transcriptional regulation of the mouse IL-7 receptor alpha promoter by glucocorticoid receptor.

Expression of the IL-7R alpha-chain (IL-7Ralpha) is strictly regulated during the development and maturation of lymphocytes. Glucocorticoids (GC) have pleiotypic effects on the growth and function of lymphocytes. Although GC have been reported to induce the transcription of IL-7Ralpha gene in human T cells, its molecular mechanism is largely unknown. In this study, we show that GC up-regulate the levels of IL-7Ralpha mRNA and protein in mouse T cells. This effect does not require protein synthesis de novo, because protein synthesis inhibitors do not block the process. Mouse IL-7Ralpha promoter has striking homology with human and rat, containing consensus motifs of Ikaros, PU.1, and Runx1 transcription factors. In addition, a conserved noncoding sequence (CNS) of approximately 270 bp was found 3.6-kb upstream of the promoter, which was designated as CNS-1. A GC receptor (GR) motif is present in the CNS-1 region. Importantly, we show by reporter assay that the IL-7Ralpha promoter has specific transcription activity in T cells. This activity highly depends on the PU.1 motif. Furthermore, GC treatment augments the transcriptional activity through the GR motif in the CNS-1 region. We also demonstrate that GR binds to the GR motif by EMSA. In addition, by chromatin immunoprecipitation assay, we show that GR is rapidly recruited to endogenous CNS-1 chromatin after GC stimulation. These results demonstrate that GR binds to the GR motif in the CNS-1 region after GC stimulation and then activates the transcription of the IL-7Ralpha promoter. Thus, this study identifies the IL-7Ralpha CNS-1 region as a GC-responsive element.

Transcriptional regulation of the mouse interleukin-2 receptor beta chain gene by Ets and Egr-1.

To clarify the mechanisms and factors involved in the regulation of mouse IL-2Rbeta gene expression, we isolated the 5'-flanking region of IL-2Rbeta gene and investigated the promoter activity. Here we elucidated the positive regulatory regions, the most potent of which are located between -50 to -30bp and -164 to -135bp. These regions contain a potentially functional Ets and Egr-1-binding sites whose mutations abrogate promoter activity. Data from electrophoretic mobility shift assay indicate that Ets and Egr-1, but not Sp1, bind to the positive regulatory regions, -50 to -30bp and -164 to -135bp, respectively. Furthermore, recruitment of Ets and Egr-1 at endogenous IL-2Rbeta promoter segments in an IL-2-dependent F7 cells was verified by the chromatin immunoprecipitation assay. This study for the first time delineates the molecular mechanisms underlying regulation of mouse IL-2Rbeta gene transcription by Ets family proteins, partially with Egr-1, and thereby further elucidates the molecular basis of lymphocyte activation and differentiation.

Seeding of dendritic epidermal T cells in the neonatal skin is reduced in 129 strain of mice.

Precursors for Thy-1(+) dendritic epidermal T cells (DETC) develop as Vgamma3(+) T cells in the fetal thymus and become distributed in the adult skin. DETC are variably distributed from site to site and from strain to strain. To elucidate the basis of strain variation, we first compared the density of DETC in the ear epidermis among different mouse strains. In the ear epidermis, we detected the highest level of DETC in C57BL/6 mice, intermediate levels in C3H and CBA/J mice, and the lowest levels in other strains including BALB/c and 129 mice. Although BALB/c and 129+Ter/Sv mice showed higher levels of DETC in the abdomen than in the ear, the levels were significantly lower than C57BL/6 mice. Furthermore, in neonatal abdominal epidermis we detected considerably lower numbers of DETC in BALB/c and 129+Ter/Sv mice than in C57BL/6 mice. In contrast, Vgamma3(+) DETC precursors in the fetal thymus are rather increased in 129+Ter/Sv mice. These results suggest that fewer DETC precursors are seeded in the neonatal skin of BALB/c and 129+Ter/Sv mice and that their expansion in the skin during neonatal to adult stages does not reach the levels in C57BL/6 mice.