Immune regulation and therapeutic application of T regulatory cells in liver diseases.

CD4+ CD25+ FOXP3+ T regulatory cells (Tregs) are a subset of the immunomodulatory cell population that can inhibit both innate and adaptive immunity by various regulatory mechanisms. In hepatic microenvironment, proliferation, plasticity, migration, and function of Tregs are interrelated to the remaining immune cells and their secreted cytokines and chemokines. In normal conditions, Tregs protect the liver from inflammatory and auto-immune responses, while disruption of this crosstalk between Tregs and other immune cells may result in the progression of chronic liver diseases and the development of hepatic malignancy. In this review, we analyze the deviance of this protective nature of Tregs in response to chronic inflammation and its involvement in inducing liver fibrosis, cirrhosis, and hepatocellular carcinoma. We will also provide a detailed emphasis on the relevance of Tregs as an effective immunotherapeutic option for autoimmune diseases, liver transplantation, and chronic liver diseases including liver cancer.

The immunogenic profile and immunomodulatory function of mesenchymal stromal / stem cells in the presence of Ptychotis verticillata.

Mesenchymal stromal/stem cells (MSCs) are considered to be a promising immunotherapeutic tool due to their easy accessibility, culture expansion possibilities, safety profile, and immunomodulatory properties. Although several studies have demonstrated the therapeutic effects of MSCs, their efficacy needs to be improved while also preserving their safety. It has been suggested that cell homeostasis may be particularly sensitive to plant extracts. The impact of natural compounds on immunity is thus a fascinating and growing field. Ptychotis verticillata and its bioactive molecules, carvacrol and thymol, are potential candidates for improving MSC therapeutic effects. They can be used as immunotherapeutic agents to regulate MSC functions and behavior during immunomodulation. Depending on their concentrations and incubation time, these compounds strengthened the immunomodulatory functions of MSCs while maintaining their immune-evasive profile. Incubating MSCs with carvacrol and thymol does not alter their hypoimmunogenicity, as no induction of the allogeneic immune response was observed. MSCs also showed enhanced abilities to reduce the proliferation of activated T cells. Thus, MSCs are immunologically responsive to bioactive molecules derived from PV. The bioactivity may depend on the whole phyto-complex of the oil. These findings may contribute to the development of safe and efficient immunotherapeutic MSCs by using medicinal plant-derived active molecules.

Umbilical Cord Mesenchymal Stromal/Stem Cells and Their Interplay with Th-17 Cell Response Pathway.

As a form of immunomodulatory therapeutics, mesenchymal stromal/stem cells (MSCs) from umbilical cord (UC) tissue were assessed for their dynamic interplay with the Th-17 immune response pathway. UC-MSCs were able to modulate lymphocyte response by promoting a Th-17-like profile. Such modulation depended on the cell ratio of the cocultures as well as the presence of an inflammatory setting underlying their plasticity. UC-MSCs significantly increased the expression of IL-17A and RORγt but differentially modulated T cell expression of IL-23R. In parallel, the secretion profile of the fifteen factors (IL1ß, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-22, IL-21, IL-23, IL-25, IL-31, IL-33, INF-γ, sCD40, and TNF-α) involved in the Th-17 immune response pathway was substantially altered during these cocultures. The modulation of these factors demonstrates the capacity of UC-MSCs to sense and actively respond to tissue challenges. Protein network and functional enrichment analysis indicated that several biological processes, molecular functions, and cellular components linked to distinct Th-17 signaling interactions are involved in several trophic, inflammatory, and immune network responses. These immunological changes and interactions with the Th-17 pathway are likely critical to tissue healing and may help to identify molecular targets that will improve therapeutic strategies involving UC-MSCs.

Role of epigenetics and the transcription factor Sp1 in the expression of the D prostanoid receptor 1 in human cartilage.

D prostanoid receptor 1 (DP1), a prostaglandin D2 receptor, plays a central role in the modulation of inflammation and cartilage metabolism. We have previously shown that activation of DP1 signaling downregulated catabolic responses in cultured chondrocytes and was protective in mouse osteoarthritis (OA). However, the mechanisms underlying its transcriptional regulation in cartilage remained poorly understood. In the present study, we aimed to characterize the human DP1 promoter and the role of DNA methylation in DP1 expression in chondrocytes. In addition, we analyzed the expression level and methylation status of the DP1 gene promoter in normal and OA cartilage. Deletion and site-directed mutagenesis analyses identified a minimal promoter region (-250/-120) containing three binding sites for specificity protein 1 (Sp1). Binding of Sp1 to the DP1 promoter was confirmed using electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays. Treatment with the Sp1 inhibitor mithramycin A reduced DP1 promoter activity and DP1 mRNA expression. Inhibition of DNA methylation by 5-Aza-2'-deoxycytidine upregulated DP1 expression, and in vitro methylation reduced the DP1 promoter activity. Neither the methylation status of the DP1 promoter nor the DP1 expression level were different between normal and OA cartilage. In conclusion, our results suggest that the transcription factor Sp1 and DNA methylation are important determinants of DP1 transcription regulation. They also suggest that the methylation status and expression level of DP1 are not altered in OA cartilage. These findings will improve our understanding of the regulatory mechanisms of DP1 transcription and may facilitate the development of intervention strategies involving DP1.

Epigenetic regulation of 15-lipoxygenase-1 expression in human chondrocytes by promoter methylation.

OBJECTIVE AND DESIGN: 15-Lipoxygenase-1 (15-LOX-1) catalyzes the biosynthesis of many anti-inflammatory and immunomodulatory lipid mediators and was reported to have protective properties in several inflammatory conditions, including osteoarthritis (OA). This study was designed to evaluate the expression of 15-LOX-1 in cartilage from normal donors and patients with OA, and to determine whether it is regulated by DNA methylation. METHODS: Cartilage samples were obtained at autopsy from normal knee joints and from OA-affected joints at the time of total knee joint replacement surgery. The expression of 15-LOX-1 was evaluated using real-time polymerase chain reaction (PCR). The role of DNA methylation in 15-LOX-1 expression was assessed using the DNA methyltransferase inhibitor 5-Aza-2'-desoxycytidine (5-Aza-dC). The effect of CpG methylation on 15-LOX-1 promoter activity was evaluated using a CpG-free luciferase vector. The DNA methylation status of the 15-LOX-1 promoter was determined by pyrosequencing. RESULTS: Expression of 15-LOX-1 was upregulated in OA compared to normal cartilage. Treatment with 5-Aza-dC increased 15-LOX-1 mRNA levels in chondrocytes, and in vitro methylation decreased 15-LOX-1 promoter activity. There was no difference in the methylation status of the 15-LOX-1 gene promoter between normal and OA cartilage. CONCLUSION: The expression level of 15-LOX-1 was elevated in OA cartilage, which may be part of a repair process. The upregulation of 15-LOX-1 in OA cartilage was not associated with the methylation status of its promoter, suggesting that other mechanisms are involved in its upregulation.

The Medicinal Potential of Mesenchymal Stem/Stromal Cells in Immuno- and Cancer Therapy.

Cancer is a highly lethal disease that causes millions of deaths worldwide, thus representing a major public health challenge [...].

Human CD4+CD25+CD127lowFOXP3+ regulatory T lymphocytes and CD4+CD25-FOXP3- conventional T lymphocytes: a differential transcriptome profile.

CD4+CD25+ FOXP3+ regulatory T cells (Tregs) represent a subpopulation of CD4+ T cells central for the suppression of physiological and pathological immune reactions. Although distinct cell surface antigens are expressed in regulatory T cells, those components are also present on the surface of activated CD4+CD25- FOXP3-T cells, thus making the discrimination between Tregs and conventional CD4+ T difficult and isolation of Tregs complex. Yet, the molecular components driving Tregs' function are still not fully characterized. Aiming at unraveling molecular components specifically marking Tregs, and upon using quantitative real-time PCR (qRT-PCR) followed by bioinformatics analysis, we identified, in this study, differential transcriptional profiles, in peripheral blood CD4 + CD25 + CD127low FOXP3+ Tregs versus CD4 + CD25-FOXP3- conventional T cells, for set of genes with distinct immunological roles. In conclusion, this study identifies some novel genes that appeared to be differentially transcribed in CD4+ Tregs versus conventional T cells. The identified genes could serve as novel molecular targets relevant to Tregs' function and isolation.

Mesenchymal Stem/Stromal Cells as a Therapeutic Tool in Cell-Based Therapy and Regenerative Medicine: An Introduction Expertise to the Topical Collection.

We are pleased to present this opening editorial, introducing our topical collection, "The New Era of Mesenchymal Stromal/Stem Cell Functional Application: State of the Art, Therapeutic Challenges and Future Directions" [...].

Fundamental and Applied Advances in Stem Cell Therapeutic Research.

We are pleased to present this Special Issue of Cells, entitled 'Feature Papers in Stem Cells' [...].

Bioscreening and pre-clinical evaluation of the impact of bioactive molecules from Ptychotis verticillata on the multilineage potential of mesenchymal stromal cells towards immune- and inflammation-mediated diseases.

OBJECTIVE AND DESIGN: Mesenchymal stromal cells (MSCs) are currently used in cell reparative medicine due to their trophic and ant-inflammatory properties. The modulation of stem cell properties by phytochemicals has been suggested as a tool to empower their tissue repair capacity. In vitro, MSCs are characterized by their tri-lineage potential that holds great interest for tissue regeneration. Ptychotis Verticillata (PV), an aromatic and medicinal plant, may be thus used to modulate the in vitro multilineage potential of MSCs. MATERIALS AND METHODS: We screened the impact of PV-derived essential oil and their bioactive molecules (thymol and carvacrol) on the in vitro multilineage potential of MSCs. Different concentrations and incubation times of these compounds were assessed during the osteogenesis and adipogenesis of MSCs. RESULTS: The analysis of 75 conditions indicates that these compounds are biologically active by promoting two major differentiation lineages from MSCs. In a time- and dose-dependent manner, thymol and carvacrol increased the osteogenesis and adipogenesis. CONCLUSION: According to these preliminary observations, the addition of PV extract may stimulate the tissue regenerative and repair functions of MSCs. Further optimization of compound extraction and characterization from PV as well as cell treatment conditions should increase their therapeutic value in combination with MSCs.

In Vitro Cellular and Molecular Interplay between Human Foreskin-Derived Mesenchymal Stromal/Stem Cells and the Th17 Cell Pathway.

Foreskin, considered a biological waste material, has been shown to be a reservoir of therapeutic cells. The immunomodulatory properties of mesenchymal stromal/stem cells (MSCs) from the foreskin (FSK-MSCs) are being evaluated in cell-based therapy for degenerative, inflammatory and autoimmune disorders. Within the injured/inflamed tissue, proinflammatory lymphocytes such as IL-17-producing T helper cells (Th17) may interact with the stromal microenvironment, including MSCs. In this context, MSCs may encounter different levels of T cells as well as specific inflammatory signals. Uncovering the cellular and molecular changes during this interplay is central for developing an efficient and safe immunotherapeutic tool. To this end, an in vitro human model of cocultures of FSK-MSCs and T cells was established. These cocultures were performed at different cell ratios in the presence of an inflammatory setting. After confirming that FSK-MSCs respond to ISCT criteria by showing a typical phenotype and multilineage potential, we evaluated by flow cytometry the expression of Th17 cell markers IL-17A, IL23 receptor and RORγt within the lymphocyte population. We also measured 15 human Th17 pathway-related cytokines. Regardless of the T cell/MSC ratio, we observed a significant increase in IL-17A expression associated with an increase in IL-23 receptor expression. Furthermore, we observed substantial modulation of IL-1ß, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, INF-γ, sCD40, and TNF-α secretion. These findings suggest that FSK-MSCs are receptive to their environment and modulate the T cell response accordingly. The changes within the secretome of the stromal and immune environment are likely relevant for the therapeutic effect of MSCs. FSK-MSCs represent a valuable cellular product for immunotherapeutic purposes that needs to be further clarified and developed.

The Therapeutic Potential of Mesenchymal Stromal Cells for Regenerative Medicine: Current Knowledge and Future Understandings.

In recent decades, research on the therapeutic potential of progenitor cells has advanced considerably. Among progenitor cells, mesenchymal stromal cells (MSCs) have attracted significant interest and have proven to be a promising tool for regenerative medicine. MSCs are isolated from various anatomical sites, including bone marrow, adipose tissue, and umbilical cord. Advances in separation, culture, and expansion techniques for MSCs have enabled their large-scale therapeutic application. This progress accompanied by the rapid improvement of transplantation practices has enhanced the utilization of MSCs in regenerative medicine. During tissue healing, MSCs may exhibit several therapeutic functions to support the repair and regeneration of injured tissue. The process underlying these effects likely involves the migration and homing of MSCs, as well as their immunotropic functions. The direct differentiation of MSCs as a cell replacement therapeutic mechanism is discussed. The fate and behavior of MSCs are further regulated by their microenvironment, which may consequently influence their repair potential. A paracrine pathway based on the release of different messengers, including regulatory factors, chemokines, cytokines, growth factors, and nucleic acids that can be secreted or packaged into extracellular vesicles, is also implicated in the therapeutic properties of MSCs. In this review, we will discuss relevant outcomes regarding the properties and roles of MSCs during tissue repair and regeneration. We will critically examine the influence of the local microenvironment, especially immunological and inflammatory signals, as well as the mechanisms underlying these therapeutic effects. Importantly, we will describe the interactions of local progenitor and immune cells with MSCs and their modulation during tissue injury. We will also highlight the crucial role of paracrine pathways, including the role of extracellular vesicles, in this healing process. Moreover, we will discuss the therapeutic potential of MSCs and MSC-derived extracellular vesicles in the treatment of COVID-19 (coronavirus disease 2019) patients. Overall, this review will provide a better understanding of MSC-based therapies as a novel immunoregenerative strategy.

Apoptotic and Non-Apoptotic Modalities of Thymoquinone-Induced Lymphoma Cell Death: Highlight of the Role of Cytosolic Calcium and Necroptosis.

Targeting non-apoptotic modalities might be therapeutically promising in diffuse large B cell lymphoma (DLBCL) patients with compromised apoptotic pathways. Thymoquinone (TQ) has been reported to promote apoptosis in cancer cells, but little is known about its effect on non-apoptotic pathways. This work investigates TQ selectivity against DLBCL cell lines and the cell death mechanisms. TQ reduces cell viability and kills cell lines with minimal toxicity on normal hematological cells. Mechanistically, TQ promotes the mitochondrial caspase pathway and increases genotoxicity. However, insensitivity of most cell lines to caspase inhibition by z-VAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone) pointed to a critical role of non-apoptotic signaling. In cells dying through non-apoptotic death, TQ increases endoplasmic reticulum (ER) stress markers and substantially increases cytosolic calcium ([Ca2+]c) through ER calcium depletion and activation of store-operated calcium entry (SOCE). Chelation of [Ca2+]c, but not SOCE inhibitors, reduces TQ-induced non-apoptotic cell death, highlighting the critical role of calcium in a non-apoptotic effect of TQ. Investigations showed that TQ-induced [Ca2+]c signaling is primarily initiated by necroptosis upstream to SOCE, and inhibition necroptosis by necrostatin-1 alone or with z-VAD-fmk blocks the cell death. Finally, TQ exhibits an improved selectivity profile over standard chemotherapy agents, suggesting a therapeutic relevance of the pro-necroptotic effect of TQ as a fail-safe mechanism for DLBCL therapies targeting apoptosis.

Transcriptional Profile of Cytokines, Regulatory Mediators and TLR in Mesenchymal Stromal Cells after Inflammatory Signaling and Cell-Passaging.

Adult human subcutaneous adipose tissue (AT) harbors a rich population of mesenchymal stromal cells (MSCs) that are of interest for tissue repair. For this purpose, it is of utmost importance to determine the response of AT-MSCs to proliferative and inflammatory signals within the damaged tissue. We have characterized the transcriptional profile of cytokines, regulatory mediators and Toll-like receptors (TLR) relevant to the response of MSCs. AT-MSCs constitutively present a distinct profile for each gene and differentially responded to inflammation and cell-passaging. Inflammation leads to an upregulation of IL-6, IL-8, IL-1ß, TNFα and CCL5 cytokine expression. Inflammation and cell-passaging increased the expression of HGF, IDO1, PTGS1, PTGS2 and TGFß. The expression of the TLR pattern was differentially modulated with TLR 1, 2, 3, 4, 9 and 10 being increased, whereas TLR 5 and 6 downregulated. Functional enrichment analysis demonstrated a complex interplay between cytokines, TLR and regulatory mediators central for tissue repair. This profiling highlights that following a combination of inflammatory and proliferative signals, the sensitivity and responsive capacity of AT-MSCs may be significantly modified. Understanding these transcriptional changes may help the development of novel therapeutic approaches.

Mesenchymal Stem/Stromal Cell Therapeutic Features: The Bridge between the Bench and the Clinic.

Mesenchymal stem/stromal cells (MSCs) are considered a relevant therapeutic product for various clinical applications [...].

Immuno-comparative screening of adult-derived human liver stem/progenitor cells for immune-inflammatory-associated molecules.

OBJECTIVE: One of the main challenges in liver cell therapy is the replacement of damaged cells and the induction of a tolerogenic microenvironment to promote graft acceptance by the recipient. Adult-derived human liver stem/progenitor cells (ADHLSCs) are currently evaluated at the clinical levels as a promising pro-regenerative and immune-modulatory tool. The expression profile of several immunological molecules may influence the local immune-inflammatory response and, therefore, modulate the tissue healing process. To increase the quality and safety of ADHLSCs before transplantation requires an appropriate analysis and characterization of their pattern expression of immune-inflammatory-associated molecules. METHODS: The expression of 27 molecules belonging to T-cell co-stimulatory pathway, CD47 partners, Ikaros family, CD300 family and TNF family were analyzed using flow cytometry. We compared their expression profiles to PBMCs, hepatocytes and ADHLSCs in both expansion and after hepatogenic differentiation culture conditions. RESULTS: This original immuno-comparative screening revealed that liver cell populations do not constitutively present significant immunological pattern compared to PBMCs. Moreover, our findings highlight that neither the expansion nor the hepatogenic differentiation induces the expression of immune-inflammatory molecules. The detailed expression characteristics (percentage of positive cells and median fluorescence intensity) of each molecule were analyzed and presented. CONCLUSION: By analyzing 27 relevant molecules, our immuno-comparative screening demonstrates that ADHLSCs keep a non-immunogenic profile independent of their expansion or hepatogenic differentiation state. Accordingly, the immunological profile of ADHLSCs seems to support their safe and efficient use in liver tissue therapeutic repair strategy.

New Anti-Leukemic Effect of Carvacrol and Thymol Combination through Synergistic Induction of Different Cell Death Pathways.

Acute myeloid leukemia (AML) is a cancer of the myeloid lineage of blood cells, and treatment for AML is lengthy and can be very expensive. Medicinal plants and their bioactive molecules are potential candidates for improving human health. In this work, we studied the effect of Ptychotis verticillata (PV) essential oil and its derivatives, carvacrol and thymol, in AML cell lines. We demonstrated that a combination of carvacrol and thymol induced tumor cell death with low toxicity on normal cells. Mechanistically, we highlighted that different molecular pathways, including apoptosis, oxidative, reticular stress, autophagy, and necrosis, are implicated in this potential synergistic effect. Using quantitative RT-PCR, Western blotting, and apoptosis inhibitors, we showed that cell death induced by the carvacrol and thymol combination is caspase-dependent in the HL60 cell line and caspase-independent in the other cell lines tested. Further investigations should focus on improving the manufacturing of these compounds and understanding their anti-tumoral mechanisms of action. These efforts will lead to an increase in the efficiency of the oncotherapy strategy regarding AML.

Therapeutic Mesenchymal Stem/Stromal Cells: Value, Challenges and Optimization.

Cellular therapy aims to replace damaged resident cells by restoring cellular and molecular environments suitable for tissue repair and regeneration. Among several candidates, mesenchymal stem/stromal cells (MSCs) represent a critical component of stromal niches known to be involved in tissue homeostasis. In vitro, MSCs appear as fibroblast-like plastic adherent cells regardless of the tissue source. The therapeutic value of MSCs is being explored in several conditions, including immunological, inflammatory and degenerative diseases, as well as cancer. An improved understanding of their origin and function would facilitate their clinical use. The stemness of MSCs is still debated and requires further study. Several terms have been used to designate MSCs, although consensual nomenclature has yet to be determined. The presence of distinct markers may facilitate the identification and isolation of specific subpopulations of MSCs. Regarding their therapeutic properties, the mechanisms underlying their immune and trophic effects imply the secretion of various mediators rather than direct cellular contact. These mediators can be packaged in extracellular vesicles, thus paving the way to exploit therapeutic cell-free products derived from MSCs. Of importance, the function of MSCs and their secretome are significantly sensitive to their environment. Several features, such as culture conditions, delivery method, therapeutic dose and the immunobiology of MSCs, may influence their clinical outcomes. In this review, we will summarize recent findings related to MSC properties. We will also discuss the main preclinical and clinical challenges that may influence the therapeutic value of MSCs and discuss some optimization strategies.

Identification of Acute Myeloid Leukemia Bone Marrow Circulating MicroRNAs.

BACKGROUND: In addition to their roles in different biological processes, microRNAs in the tumor microenvironment appear to be potential diagnostic and prognostic biomarkers for various malignant diseases, including acute myeloid leukemia (AML). To date, no screening of circulating miRNAs has been carried out in the bone marrow compartment of AML. Accordingly, we investigated the circulating miRNA profile in AML bone marrow at diagnosis (AMLD) and first complete remission post treatment (AMLPT) in comparison to healthy donors (HD). METHODS: Circulating miRNAs were isolated from AML bone marrow aspirations, and a low-density TaqMan miRNA array was performed to identify deregulated miRNAs followed by quantitative RT-PCR to validate the results. Bioinformatic analysis was conducted to evaluate the diagnostic and prognostic accuracy of the highly and significantly identified deregulated miRNA(s) as potential candidate biomarker(s). RESULTS: We found several deregulated miRNAs between the AMLD vs. HD vs. AMLPT groups, which were involved in tumor progression and immune suppression pathways. We also identified significant diagnostic and prognostic signatures with the ability to predict AML patient treatment response. CONCLUSIONS: This study provides a possible role of enriched circulating bone marrow miRNAs in the initiation and progression of AML and highlights new markers for prognosis and treatment monitoring.

Impact of Bone Marrow miR-21 Expression on Acute Myeloid Leukemia T Lymphocyte Fragility and Dysfunction.

BACKGROUND: Acute myeloid leukemia (AML) is a hematopoietic malignancy in which antitumor immunity is impaired. The therapeutic management of AML requires understanding the mechanisms involved in the fragility and immune dysfunction of AML T lymphocytes. METHODS: In this study, T lymphocytes from healthy donors (HD) and AML patients were used. Extracellular vesicles (EVs) from leukemic cells were screened for their microRNA content and impact on T lymphocytes. Flow cytometry, transcriptomic as well as lentiviral transduction techniques were used to carry out the research. RESULTS: We observed increased cell death of T lymphocytes from AML patients. EVs from leukemia myeloid cell lines harbored several miRNAs, including miR-21, and were able to induce T lymphocyte death. Compared to that in HD, miR-21 was overexpressed in both the bone marrow fluid and infiltrating T lymphocytes of AML patients. MiR-21 induces T lymphocyte cell death by upregulating proapoptotic gene expression. It also increases the immunosuppressive profile of T lymphocytes by upregulating the IL13, IL4, IL10, and FoxP3 genes. CONCLUSIONS: Our results demonstrate that miR-21 plays a significant role in AML T lymphocyte dysfunction and apoptosis. Targeting miR-21 may be a novel approach to restore the efficacy of the immune response against AML.