IFN-γ-Licensed Mesenchymal Stem Cells Are More Susceptible to Death when Exposed to Quorum-Sensing Signal Molecule OdDHL and Less Effective in Inhibiting the Growth of Pseudomonas aeruginosa.

Currently, a series of licensing strategies has been investigated to enhance the functional properties of mesenchymal stem cells (MSCs). Licensing with IFN-γ is one of the most investigated strategies for enhancing the immunosuppressive potential of such cells. However, it is not yet known whether this licensing strategy could interfere with the ability of MSCs to control bacterial growth, which may be relevant considering their clinical potential. In this study, we compared the antimicrobial potential of IFN-γ-licensed and unlicensed MSCs by exposing them to Pseudomonas aeruginosa and its quorum-sensing inducer molecule OdDHL. Our data show that-when challenged with OdDHL-IFN-γ-licensed and unlicensed MSCs present increased levels of the antimicrobial HAMP transcript, but that only IFN-γ-licensed MSCs undergo modulation of CASP1 and BCL2, entering apoptosis. Furthermore, we demonstrate that only IFN-γ-licensed MSCs show modulation in genes involved in apoptosis and tend to undergo cell death when cultured with P. aeruginosa. As a consequence, IFN-γ-licensed MSCs showed lower capacity to control bacterial growth, compared to unlicensed MSCs. Taken together, our observations reveal an increased susceptibility to apoptosis of IFN-γ-licensed MSCs, which compromises their potential to control the bacterial growth in vitro. These findings are relevant to the field of cell therapy, considering the potential applicability of MSCs.

Characterization of ibrutinib's effects on the morphology, proliferation, phenotype, viability, and anti-inflammatory potential of adipose-derived mesenchymal stromal cells.

Ibrutinib (IB) is a tyrosine kinase inhibitor (TKI) that has immunomodulatory action and can be used as second-line therapy for steroid-refractory or steroid-resistant chronic Graft versus Host Disease (cGVHD). Mesenchymal stromal cells (MSCs) are distributed throughout the body and their infusion has also been explored as a second-line therapeutic alternative for the treatment of cGVHD. Considering the currently unknown effects of IB on endogenous MSCs, as well as the possible combined use of IB and MSCs for cGVHD, we investigated whether adipose tissue-derived MSCs present IB-targets, as well as the consequences of treating MSCs with this drug, regarding cell viability, proliferation, phenotype, and anti-inflammatory potential. Interestingly, we show for the first time that MSCs express several IB target genes. Also of note, the treatment of such cells with this TKI elevated the levels of CD90 and CD105 surface proteins, as well as VCAM-1. Furthermore, IB-treated MSCs presented increased mRNA expression of the anti-inflammatory genes PD-L1, TSG-6, and IL-10. However, continued exposure to IB, even at low doses, compromised the viability of MSCs. These data indicate that the use of IB can stimulate an anti-inflammatory profile in MSCs, but also that a continued exposure to IB can compromise MSC viability over time.

GLP and G9a histone methyltransferases as potential therapeutic targets for lymphoid neoplasms.

Histone methyltransferases (HMTs) are enzymes that regulate histone methylation and play an important role in controlling transcription by altering the chromatin structure. Aberrant activation of HMTs has been widely reported in certain types of neoplastic cells. Among them, G9a/EHMT2 and GLP/EHMT1 are crucial for H3K9 methylation, and their dysregulation has been associated with tumor initiation and progression in different types of cancer. More recently, it has been shown that G9a and GLP appear to play a critical role in several lymphoid hematologic malignancies. Importantly, the key roles played by both enzymes in various diseases made them attractive targets for drug development. In fact, in recent years, several groups have tried to develop small molecule inhibitors targeting their epigenetic activities as potential anticancer therapeutic tools. In this review, we discuss the physiological role of GLP and G9a, their oncogenic functions in hematologic malignancies of the lymphoid lineage, and the therapeutic potential of epigenetic drugs targeting G9a/GLP for cancer treatment.

Senescence on Dental Pulp Cells: Effects on Morphology, Migration, Proliferation, and Immune Response.

INTRODUCTION: Evidence indicates that senescence can affect essential dental pulp functions, such as defense capacity and repair, consequently affecting the successes of conservative endodontic treatments. This study aims to evaluate the effects of senescence on the morphology, migration, proliferation, and immune response of human dental pulp cells. METHODS: Cells were treated with doxorubicin to induce senescence, confirmed by ß-galactosidase staining. Morphological changes, cellular proliferation, and migration were evaluated by scanning electron microscopy, trypan blue cells, and the scratch method, respectively. Modifications in the immune response were evaluated by measuring the genes for pro-inflammatory cytokines tumor necrosis factor alpha and interleukin (IL)-6 and anti-inflammatory cytokines transforming growth factor beta 1 and IL-10 using the real time polymerase chain reaction assay. RESULTS: An increase in cell size and a decrease in the number of extensions were observed in senescent cells. A reduction in the proliferative and migratory capacity was also found in senescent cells. In addition, there was an increase in the gene expression of inflammatory cytokines tumor necrosis factor alpha and IL-6 and a decrease in the gene expression of IL-10 and transforming growth factor beta-1, suggesting an exacerbated inflammatory situation associated with immunosuppression. CONCLUSIONS: Cellular senescence is possibly a condition that affects prognoses of conservative endodontic treatments, as it affects primordial cellular functions related to this treatment.

The Peptide Salamandrin-I Modulates Components Involved in Pyroptosis and Induces Cell Death in Human Leukemia Cell Line HL-60.

Amphibian secretions have been extensively investigated for the production of bioactive molecules. Salamandrin-I is an antioxidant peptide, isolated from the skin secretion of the fire salamander, that has induced no toxicity in microglia or erythrocytes. Importantly, the administration of antioxidants may constitute an adequate therapeutic approach to cancer treatment. Here, with the purpose of better characterizing the therapeutic potential of salamandrin-I, we investigated whether this antioxidant peptide also exerts anticancer activity, using the human leukemia cell line HL-60 as a cancer model. Salamandrin-I treatment induced a significant reduction in HL-60 proliferation, which was accompanied by cell cycle arrest. Furthermore, the peptide-induced cell death showed a significant increase in the LDH release in HL-60 cells. The cellular toxicity exerted by salamandrin-I is possibly related to pyroptosis, since the HL-60 cells showed loss of mitochondrial membrane potential and hyperexpression of inflammasome components following the peptide treatment. This is the first demonstration of the anticancer potential of the salamandrin-I peptide. Such results are important, as they offer relevant insights into the field of cancer therapy and allow the design of future bioactive molecules using salamandrin-I as a template.

Regulatory T-Cell Enhancement, Expression of Adhesion Molecules, and Production of Anti-Inflammatory Factors Are Differentially Modulated by Spheroid-Cultured Mesenchymal Stem Cells.

The culture of mesenchymal stem cells (MSCs) as spheroids promotes a more physiological cellular behavior, as it more accurately reflects the biological microenvironment. Nevertheless, mixed results have been found regarding the immunosuppressive properties of spheroid-cultured MSCs (3D-MSCs), the mechanisms of immunoregulation of 3D-MSCs being scarcely described at this point. In the present study, we constructed spheroids from MSCs and compared their immunosuppressive potential with that of MSCs cultured in monolayer (2D-MSCs). First, we evaluated the ability of 2D-MSCs and 3D-MSCs to control the activation and proliferation of T-cells. Next, we evaluated the percentage of regulatory T-cells (Tregs) after the co-culturing of peripheral blood mononuclear cells (PBMCs) with 2D-MSCs and 3D-MSCs. Finally, we investigated the expression of adhesion molecules, as well as the expressions of several anti-inflammatory transcripts in 2D-MSCs and 3D-MSCs maintained in both inflammatory and non-inflammatory conditions. Interestingly, our data show that several anti-inflammatory genes are up-regulated in 3D-MSCs, and that these cells can control T-cell proliferation. Nevertheless, 2D-MSCs are more efficient in suppressing the immune cell proliferation. Importantly, contrary to what was observed in 3D-MSCs, the expressions of ICAM-1 and VCAM-1 are significantly upregulated in 2D-MSCs exposed to an inflammatory environment. Furthermore, only 2D-MSCs are able to promote the enhancement of Tregs. Taken together, our data clearly show that the immunosuppressive potential of MSCs is significantly impacted by their shape, and highlights the important role of cell-cell adhesion molecules for optimal MSC immunomodulatory function.

Ezrin is highly expressed and a druggable target in chronic lymphocytic leukemia.

AIMS: Despite the development of therapeutic strategies for chronic lymphocytic leukemia (CLL), most patients remain incurable, relapse, or refractory to current treatments, indicating the need to expand the antineoplastic repertoire for this disease. Ezrin (EZR) is a known oncogene in solid tumors and plays a key role in cell survival and BCR-mediated signaling activation in B-cell lymphomas. However, its role in hematological neoplasms remains poorly explored. MAIN METHODS: The present study assessed EZR expression in samples from CLL patients and healthy donors and evaluated the cellular and molecular effects of a pharmacological EZR inhibitor, NSC305787, in CLL cellular models. KEY FINDINGS: EZR was highly expressed and positively associated with relevant signaling pathways related to CLL development and progression, including TP53, PI3K/AKT/mTOR, NF-κB, and MAPK. NSC305787 reduced viability, clonogenicity, and cell cycle progression and induced apoptosis in CLL cells. Pharmacological EZR inhibition also attenuated ERK, S6RP, and NF-κB activation, indicating that EZR not only associates with but also activates these signaling pathways in CLL. Ex vivo assays revealed that the EZR inhibition-induced cell viability reduction was independent of molecular risk and the Binet stage. SIGNIFICANCE: Our study provides insights into EZR as a pharmacological target in CLL, shedding light on a novel strategy for treating this disease.

The Inflammatory Status of Soluble Microenvironment Influences the Capacity of Melanoma Cells to Control T-Cell Responses.

The development of immunotherapeutic approaches for the treatment of melanoma requires a better understanding of immunoescape mechanisms of tumor cells and how they interact with other tumor-resident cell types. Here, we evaluated how the conditioned media of resting (rCM) and immune-activated PBMCs (iCM) influence the ability of a metastatic melanoma cell line (MeWo) to control T-cells function. MeWo cells were expanded in RPMI, rCM, or iCM and the secretome generated after cell expansion was identified as MeSec (RPMI), niSec (non-inflammatory), or iSec (inflammatory secretome), respectively. Then, the immunomodulatory potential of such secretomes was tested in PHA-activated PBMCs. iCM induced higher levels of IFN-γ and IL-10 in treated melanoma cells compared to rCM, as well as higher IDO and PD-L1 expression. The iSec was able to inhibit T-cell activation and proliferation. Interestingly, PBMCs treated with iSec presented a reduced expression of the regulators of Th1 and Th2 responses T-BET and GATA-3, as well as low expression of IFN-γ, and co-stimulatory molecules TIM-3 and LAG-3. Importantly, our findings show that melanoma may benefit from an inflammatory microenvironment to enhance its ability to control the T-cell response. Interestingly, such an immunomodulatory effect involves the inhibition of the checkpoint molecules LAG-3 and TIM-3, which are currently investigated as important therapeutic targets for melanoma treatment. Further studies are needed to better understand how checkpoint molecules are modulated by paracrine and cell contact-dependent interaction between melanoma and immune cells. Such advances are fundamental for the development of new therapeutic approaches focused on melanoma immunotherapy.

Differential peripheral blood mononuclear cell reactivity against SARS-CoV-2 proteins in naïve and previously infected subjects following COVID-19 vaccination.

The decline in vaccine efficacy and the risk of reinfection by SARS-CoV-2 make new studies important to better characterize the immune response against the virus and its components. Here, we investigated the pattern of activation of T-cells and the expression of inflammatory factors by PBMCs obtained from naïve and previously infected subjects following COVID-19 vaccination, after PBMCs stimulation with S1, RBD, and N-RBD SARS-CoV-2 proteins. PBMCs showed low levels of ACE2 and TMPRSS2 transcripts, which were not modulated by the exposure of these cells to SARS-CoV-2 proteins. Compared to S1 and RBD, N-RBD stimulation showed a greater ability to stimulate T-cell reactivity, according to CD25 and CD69 markers. Interestingly, T-cell reactivity was more pronounced in vaccinated subjects with prior SARS-CoV-2 infection than in vaccinated donors who never had been diagnosed with COVID-19. Finally, N-RBD stimulation promoted greater expression of IL-6 and IFN-γ in PBMCs, which reinforces the greater immunogenic potential of this protein in the vaccinated subjects. These data suggest that PBMCs from previously infected and vaccinated subjects are more reactive than those derived from just vaccinated donors. Moreover, the N-RBD together viral proteins showed a greater stimulatory capacity than S1 and RBD viral proteins.

Dissecting the relationship between antimicrobial peptides and mesenchymal stem cells.

Among the various biological properties presented by Mesenchymal Stem Cells (MSCs), their ability to control the immune response and fight pathogen infection through the production of antimicrobial peptides (AMPs) have been the subject of intense research in recent years. AMPs secreted by MSCs exhibit activity against a wide range of microorganisms, including bacteria, fungi, yeasts, and viruses. The main AMPs produced by these cells are hepcidin, cathelicidin LL-37, and ß-defensin-2. In addition to acting against pathogens, those AMPs have also been shown to interact with MSCs to modulate MSC proliferation, migration, and regeneration, indicating that such peptides exert a more diverse biological effect than initially thought. In the present review, we discuss the production of AMPs by MSCs, revise the multiple functions of these peptides, including their influence over MSCs, and present an overview of clinical situations in which the antimicrobial properties of MSCs may be explored for therapy. Finally, we discuss possibilities of combining MSCs and AMPs to generate improved therapeutic strategies.

Epigenetic priming by EHMT1/EHMT2 in acute lymphoblastic leukemia induces TP53 and TP73 overexpression and promotes cell death.

Euchromatic histone-lysine N-methyltransferase 1 (EHMT1) and EHMT2 are upregulated in various human cancers, and their deregulation is associated with tumor development and progression. In this paper, we investigated the expression level of EHMT1/EHMT2 in acute lymphoblastic leukemia (ALL) and whether the modulation of these enzymes could have any cellular or molecular impact on ALL cells. For this, we used UNC0646 as a priming strategy to target EHMT1/EHMT2 and investigated its effect on proliferation and cell viability of Jurkat cells by MTT assay. Then, considering the IC50 and IC75, cellular death was determined by Annexin V/PI staining using flow cytometry. Finally, we investigated by RT-PCR the molecular bases that could be involved in the observed effects. Interestingly, accessing the International Microarray Innovations in Leukemia (MILE) study group, we detected that both EHMT1 and EHMT2 are overexpressed in ALL. More important, we determined that inhibition of EHMT1/EHMT2 significantly decreased Jurkat cell viability in a dose-dependent manner. Accordingly, we observed that inhibition of EHMT1/EHMT2 promoted Jurkat cell death, which was accompanied by increased expression of P53, TP73, BAX, and MDM4. These results clearly indicate that inhibition of EHMT1/EHMT2 induces pro-apoptotic gene expression in ALL and promotes cell death. More importantly, the modulation of these histone methyltransferases may be a promising epigenetic target for ALL treatment.

Mesenchymal Stem Cells: A New Piece in the Puzzle of COVID-19 Treatment.

COVID-19 is a disease characterized by a strong inflammatory response in severe cases, which fails to respond to corticosteroid therapy. In the context of the current COVID-19 outbreak and the critical information gaps regarding the disease, several different therapeutic strategies are under investigation, including the use of stem cells. In the present manuscript, we provide an analysis of the rationale underlying the application of stem cells to manage COVID-19, and also a comprehensive compendium of the 69 clinical trials underway worldwide aiming to investigate the application of stem cells to treat COVID-19. Even though data are still scarce, it is already possible to observe the protagonism of China in testing mesenchymal stem cells (MSCs) for COVID-19. Furthermore, it is possible to determine that current efforts focus on the use of multiple infusions of high numbers of stem cells and derived products, as well as to acknowledge the positive results obtained by independent groups who publicized the therapeutic benefits provided by such therapies in 51 COVID-19 patients. In such a rapid-paced field, up-to-date systematic studies and meta-analysis will aid the scientific community to separate hype from hope and offer an unbiased position to the society and governments.

GVHD-derived plasma as a priming strategy of mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cell (MSC) therapy is an important alternative for GVHD treatment, but a third of patients fail to respond to such therapy. Therefore, strategies to enhance the immunosuppressive potential of MSCs constitute an active area of investigation. Here, we proposed an innovative priming strategy based on the plasma obtained from GVHD patients and tested whether this approach could enhance the immunosuppressive capacity of MSCs. METHODS: We obtained the plasma from healthy as well as acute (aGVHD) and chronic (cGVHD) GVHD donors. Plasma samples were characterized according to the TNF-α, IFN-γ, IL-10, IL-1ß, IL-12p40, and IL-15 cytokine levels. The MSCs primed with such plasmas were investigated according to surface markers, morphology, proliferation, mRNA expression, and the capacity to control T cell proliferation and Treg generation. RESULTS: Interestingly, 57% of aGVHD and 33% of cGVHD plasmas significantly enhanced the immunosuppressive potential of MSCs. The most suppressive MSCs presented altered morphology, and those primed with cGHVD displayed a pronounced overexpression of ICAM-1 on their surface. Furthermore, we observed that the ratio of IFN-γ to IL-10 cytokine levels in the plasma used for MSC priming was significantly correlated with higher suppressive potential and Treg generation induction by primed MSCs, regardless of the clinical status of the donor. CONCLUSIONS: This work constitutes an important proof of concept which demonstrates that it is possible to prime MSCs with biological material and also that the cytokine levels in the plasma may affect the MSC immunosuppressive potential, serving as the basis for the development of new therapeutic approaches for the treatment of immune diseases.

The immunosuppressive mechanisms of mesenchymal stem cells are differentially regulated by platelet poor plasma and fetal bovine serum supplemented media.

PURPOSE: Mesenchymal Stem Cells (MSCs) can interact with and modulate the functions of all immune cells, suppressing both the innate and adaptive immune responses. Currently, most of the in vitro studies which have led to the description of MSC properties have resulted from MSC culture in the presence of fetal bovine serum (FBS), in spite of the recognition of FBS limitations and attempts to substitute this component from the MSC media. METHODS: Herein, we compare FBS and Platelet Poor Plasma (PPP) as MSC media supplements, according to Adipose-derived MSC (AMSC) phenotype, proliferation and immunoregulatory mechanisms. RESULTS: Interestingly, despite maintaining the classic phenotypic profile of MSCs, PPP cultured AMSCs showed impaired proliferative potential. Furthermore, our results clearly show that AMSC culture with PPP leads to decreased expression of soluble immunosuppressive factors, which resulted in decreased capacity of inducing regulatory T-cells (Tregs) generation by these cells. In contrast, PPP supplementation promoted enhanced VCAM-1 and ICAM-1 expression on AMSC surface. Therefore, AMSCs cultured with PPP showed limited potential to produce soluble immunomodulatory factors, indicating a reduced capacity to control the immune system thought paracrine activity. CONCLUSION: Overall, our data sheds light on the importance of culture media supplementation for MSC immunomodulatory behavior, as well as serving as an alert regarding the complexity of replacing FBS in MSC culture.

Assessment of the Immunosuppressive Potential of INF-γ Licensed Adipose Mesenchymal Stem Cells, Their Secretome and Extracellular Vesicles.

There is an active search for the ideal strategy to potentialize the effects of Mesenchymal Stem-Cells (MSCs) over the immune system. Also, part of the scientific community is seeking to elucidate the therapeutic potential of MSCs secretome and its extracellular vesicles (EVs), in order to avoid the complexity of a cellular therapy. Here, we investigate the effects of human adipose MSCs (AMSCs) licensing with INF-γ and TLR3 agonist over AMSCs proliferation, migration, as well as the immunomodulatory function. Furthermore, we evaluated how the licensing of AMSCs affected the immunomodulatory function of AMSC derived-secretome, including their EVs. INF-γ licensed-AMSCs presented an elevated expression of indoleamine 2,3-dioxygenase (IDO), accompanied by increased ICAM-1, as well as a higher immunosuppressive potential, compared to unlicensed AMSCs. Interestingly, the conditioned medium obtained from INF-γ licensed-AMSCs also revealed a slightly superior immunosuppressive potential, compared to other licensing strategies. Therefore, unlicensed and INF-γ licensed-AMSCs groups were used to isolate EVs. Interestingly, EVs isolated from both groups displayed similar capacity to inhibit T-cell proliferation. EVs isolated from both groups shared similar TGF-ß and Galectin-1 mRNA content but only EVs derived from INF-γ licensed-AMSCs expressed IDO mRNA. In summary, we demonstrated that INF-γ licensing of AMSCs provides an immunosuppressive advantage both from a cell-cell contact-dependent perspective, as well as in a cell-free context. Interestingly, EVs derived from unlicensed and INF-γ licensed-AMSCs have similar ability to control activated T-cell proliferation. These results contribute towards the development of new strategies to control the immune response based on AMSCs or their derived products.

LL-37 treatment on human peripheral blood mononuclear cells modulates immune response and promotes regulatory T-cells generation.

LL-37 is a host-defense peptide (HDP) and exerts a broad spectrum of microbicidal activity against bacteria, fungi, and viral pathogens. This peptide also interacts with human cells and influences their behavior, promoting angiogenesis, wound healing, immunomodulation, and affecting apoptosis. Lately, significant advances have been achieved regarding the elucidation of underlying mechanisms related to LL-37 effects over neutrophil and monocytes. However, how T-cells respond to LL-37 stimulation is still largely unknown. Here, we used flow cytometry to evaluate the effects of LL-37 over peripheral blood mononuclear cells (PBMCs) viability, T-cell proliferation, T-cell activation, as well as the generation of regulatory T-cells (Tregs). Those aspects were assessed both in immune homeostatic and inflammatory milieu. Furthermore, we investigated the transcript levels of the inflammatory factors INF-γ, TNF-ɑ, and TGF-ß in these conditions. Interestingly, our data revealed that the treatment of PBMCs with LL-37 enhanced the viability of these cells and exerted wide effects over T cell response. Upon activation, LL-37 treated T-cells presented lower proliferation and also increased generation of Tregs. Finally, while non-stimulated cells increased the expression of inflammatory factors when treated with LL-37, activated cells treated with LL-37 presented a decreased production of the same inflammatory mediators. These results are important for the immunotherapy field, and indicate that the use of LL-37 must be carefully evaluated in both homeostatic and inflammatory scenarios, since the microenvironment clearly plays a crucial role in determining how T-cells respond to LL-37.