The downregulation of type I IFN signaling in G-MDSCs under tumor conditions promotes their development towards an immunosuppressive phenotype

Tumors modify myeloid cell differentiation and induce an immunosuppressive microenvironment. Granulocytic myeloid-derived suppressor cells (G-MDSCs), the main subgroup of myeloid-derived suppressor cells (MDSCs), are immature myeloid cells (IMCs) with immunosuppressive activity and exist in tumor-bearing hosts. The reason why these cells diverge from a normal differentiation pathway and are shaped into immunosuppressive cells remains unclear. Here, we reported that the increase of granulocyte colony-stimulating factor (G-CSF) in mouse serum with tumor progression encouraged G-MDSCs to obtain immunosuppressive traits in peripheral blood through the PI3K-Akt/mTOR pathway. Importantly, we found that downregulation of type I interferon (IFN-I) signaling in G-MDSCs was a prerequisite for their immunosuppressive effects. Suppressor of cytokine signaling (SOCS1), the action of which is dependent on IFN-I signaling, inhibited the activation of the PI3K-Akt/mTOR pathway by directly interacting with Akt, indicating that the differentiation of immunosuppressive G-MDSCs involves a transition from immune activation to immune tolerance. Our study suggests that increasing IFN-I signaling in G-MDSCs may be a strategy for reprograming immunosuppressive myelopoiesis and slowing tumor progression.

Hyperosmolarity Impairs Human Extravillous Trophoblast Differentiation by Caveolae Internalization

We recently reported that an intact caveolar structure is necessary for adequate cell migration and tubulogenesis of the human extravillous trophoblast (EVT) cells. Emerging evidence supports that hyperosmolarity induces the internalization of caveolae into the cytoplasm and accelerates their turnover. Furthermore, signaling pathways associated with the regulation of trophoblast differentiation are localized in caveolae. We hypothesized that hyperosmolarity impairs EVT differentiation and caveolae/caveolin−1 (Cav-1) participates in this process. EVT cells (Swan 71 cell line) were cultured in complete Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 and exposed to hyperosmolar condition (generated by the addition of 100 mM sucrose). Hyperosmolarity altered the EVT cell migration and the formation of tube-like structures. In addition, cell invasion was decreased along with a reduction in the latent and active forms of matrix metalloproteinase-2 (MMP−2) secreted by these cells. With respect to Cav-1 protein abundance, we found that hyperosmolarity enhanced its degradation by the lysosomal pathway. Accordingly, in the hyperosmolar condition, we also observed a significant increase in the number of vacuoles and the internalization of the caveolae into the cytoplasm. Taken together, our findings suggest that hyperosmolarity may induce caveolae internalization and increase their turnover, compromising the normal differentiation of EVT cells.

G9a: An Emerging Epigenetic Target for Melanoma Therapy

Epigenetic regulation is a crucial component of DNA maintenance and cellular identity. As our understanding of the vast array of proteins that contribute to chromatin accessibility has advanced, the role of epigenetic remodelers in disease has become more apparent. G9a is a histone methyltransferase that contributes to immune cell differentiation and function, neuronal development, and has been implicated in diseases, including cancer. In melanoma, recurrent mutations and amplifications of G9a have led to its identification as a therapeutic target. The pathways that are regulated by G9a provide an insight into relevant biomarkers for patient stratification. Future work is aided by the breadth of literature on G9a function during normal differentiation and development, along with similarities to EZH2, another histone methyltransferase that forms a synthetic lethal relationship with members of the SWI/SNF complex in certain cancers. Here, we review the literature on G9a, its role in melanoma, and lessons from EZH2 inhibitor studies.

Single-cell ATAC-seq reveals GATA2-dependent priming defect in myeloid and a maturation bottleneck in lymphoid lineages

Germline heterozygous mutations in GATA2 are associated with a syndrome characterized by cytopenias, atypical infections, and increased risk of hematologic malignancies. Here, we generated a zebrafish mutant of gata2b that recapitulated the myelomonocytopenia and B-cell lymphopenia of GATA2 deficiency syndrome. Using single-cell assay for transposase accessible chromatin with sequencing of marrow cells, we showed that loss of gata2b led to contrasting alterations in chromosome accessibility in early myeloid and lymphoid progenitors, associated with defects in gene expression. Within the myeloid lineage in gata2b mutant zebrafish, we identified an attenuated myeloid differentiation with reduced transcriptional priming and skewing away from the monocytic program. In contrast, in early lymphoid progenitors, gata2b loss led to accumulation of B-lymphoid transcription factor accessibility coupled with increased expression of the B-cell lineage-specification program. However, gata2b mutant zebrafish had incomplete B-cell lymphopoiesis with loss of lineage-specific transcription factor accessibility in differentiating B cells, in the context of aberrantly reduced oxidative metabolic pathways. Our results establish that transcriptional events in early progenitors driven by Gata2 are required to complete normal differentiation.

Flavonoid Kaempferol inhibits the Proliferation and Survival of Human Leukemia HL60 cells

Background: Acute myeloid leukemia (AML) is an aggressive type of leukemia adversely affecting the normal differentiation and proliferation process of human hematopoietic myeloid lineage. During the last decades, Kaempferol (Kae) (3,4′,5,7-tetrahydroxyflavone) is considered a flavonoid with useful medical significance, capable of inhibiting various types of leukemia (e.g., AML). Objective: To evaluate the Kae effect on the proliferation and apoptosis of a human AML cell line, HL60. Methods: The proliferation capability of the HL60 cells was estimated by MTT assay after 12, 24, and 48 hours after the exposure to Kae at a series of concentrations, including 10, 25, 50, and 75 µM. Also, the apoptosis level of HL60 cells was measured 48 hours after the exposure to various concentrations of Kae (10, 25, and 50 µM) using Annexin-V/PI staining and FACS analysis. Besides, the gene expression of CDK1/2, Bcl-2, survivin, c-FLIP, Mcl-1, XIAP, Bax, and caspase 3 and 8 was assessed following the treatment of HL60 cells with Kae (25 and 50 µM) by Real-Time PCR. Results: The anti-proliferation activity of Kae showed an ascending pattern over time and reached the maximum level after 48 hours of HL60 cells exposure with Kae. Also, it was able to trigger apoptosis of HL60 cells, in particular, at 50 µM concentration. On the other hand, Kae could modify the expression levels of the candidate’s genes in treated cells. Conclusion: The promising results of using Kae against the HL60 cells have made it a good drug candidate to treat AML through the up-regulation of caspases expression and down-regulation of anti-apoptotic proteins.

The chaperonin CCT8 controls proteostasis essential for T cell maturation, selection, and function

T cells rely for their development and function on the correct folding and turnover of proteins generated in response to a broad range of molecular cues. In the absence of the eukaryotic type II chaperonin complex, CCT, T cell activation induced changes in the proteome are compromised including the formation of nuclear actin filaments and the formation of a normal cell stress response. Consequently, thymocyte maturation and selection, and T cell homeostatic maintenance and receptor-mediated activation are severely impaired. In the absence of CCT-controlled protein folding, Th2 polarization diverges from normal differentiation with paradoxical continued IFN-γ expression. As a result, CCT-deficient T cells fail to generate an efficient immune protection against helminths as they are unable to sustain a coordinated recruitment of the innate and adaptive immune systems. These findings thus demonstrate that normal T cell biology is critically dependent on CCT-controlled proteostasis and that its absence is incompatible with protective immunity.

Liposomes embedded with differentiating factors as a new strategy for enhancing DPSC osteogenic commitment

Human dental pulp stem cell (DPSC) differentiation toward the osteoblastic phenotype is enhanced when culture media are supplemented with differentiating factors, i.e. ascorbic acid, β-glycerophosphate and dexamethasone. Liposomes, spherical vesicles formed by a phospholipid bilayer, are frequently used as carriers for drugs, growth factors and hydrophobic molecules. The aim of this work was to speed up DPSC commitment to the osteogenic lineage by embedding differentiating factors within liposomes. Firstly, liposomes were prepared by rehydrating a phospholipidic thin film and characterised in terms of dimensions. Secondly, liposome-exposed DPSCs were characterised by their immunophenotypic profile. Levels of CD90 were significantly decreased in the presence of liposomes filled with ascorbic acid, β-glycerophosphate and dexamethasone (Lipo-Mix) with respect to normal differentiation medium (DM), while CD73 and CD29 expression were enhanced, suggesting osteogenic commitment. Additionally, an appreciable extracellular matrix deposition is detected. Thirdly, the Lipo-Mix formulation better increases alkaline phosphatase activity and levels of Collagen I secretion with respect to DM. In parallel, the new liposome formulation is capable of decreasing the release of H2O2 and of triggering a precocious antioxidant cell response, redressing the redox balance required upon mesenchymal stem cell commitment to osteogenesis. It can be therefore hypothesised that Lipo-Mix could represent a suitable tool for clinical regenerative purposes in the field of tissue engineering by speeding up DPSC osteogenic commitment, mineralised matrix deposition and remodelling.

QKI is a critical pre-mRNA alternative splicing regulator of cardiac myofibrillogenesis and contractile function

The RNA-binding protein QKI belongs to the hnRNP K-homology domain protein family, a well-known regulator of pre-mRNA alternative splicing and is associated with several neurodevelopmental disorders. Qki is found highly expressed in developing and adult hearts. By employing the human embryonic stem cell (hESC) to cardiomyocyte differentiation system and generating QKI-deficient hESCs (hESCs-QKIdel) using CRISPR/Cas9 gene editing technology, we analyze the physiological role of QKI in cardiomyocyte differentiation, maturation, and contractile function. hESCs-QKIdel largely maintain normal pluripotency and normal differentiation potential for the generation of early cardiogenic progenitors, but they fail to transition into functional cardiomyocytes. In this work, by using a series of transcriptomic, cell and biochemical analyses, and the Qki-deficient mouse model, we demonstrate that QKI is indispensable to cardiac sarcomerogenesis and cardiac function through its regulation of alternative splicing in genes involved in Z-disc formation and contractile physiology, suggesting that QKI is associated with the pathogenesis of certain forms of cardiomyopathies.

Glucocorticoids induce osteonecrosis of the femoral head through the Hippo signaling pathway

Osteonecrosis of the femoral head (ONFH) induced by glucocorticoids (GCs) has been considered to be associated with the dysfunction of bone marrow mesenchymal stem cells (BMSCs). Studies have reported that GCs can regulate the normal differentiation of BMSCs. However, the exact mechanism of this regulation remains unclear. In this study, we used methylprednisolone (MPS) to induce BMSCs, and then found that the Hippo signaling pathway was upregulated in a dose-dependent manner compared to that in the control group. In addition, the osteogenic ability of BMSCs was decreased, as evaluated by Alizarin Red S staining analysis and alkaline phosphatase activity assays, accompanied by the downregulated expression of Runx2, osteopontin, and osteocalcin. Additionally, the adipogenic capacity of BMSCs under the MPS conditions was increased, as identified by Oil Red O staining with upregulated triglyceride and PPARγ expression. Moreover, suppression by knockdown of MST1 was found to attenuate the Hippo signaling pathway and adipogenic differentiation, while enhancing osteogenic differentiation. In conclusion, our findings revealed that the Hippo signaling pathway was involved in GC-ONFH by affecting the osteogenic and adipogenic differentiation capacities of BMSCs. Our study could provide a basis for further investigation of the specific function of the Hippo pathway in ONFH.

Oxidative Stress Impact on the Transcriptome of Differentiating Neuroblastoma Cells: Implication for Psychiatric Disorders

Prenatal environmental exposures that have been shown to induce oxidative stress (OS) during pregnancy, such as smoking and alcohol consumption, are risk factors for the onset of schizophrenia and other neurodevelopmental disorders (NDDs). While the OS role in the etiology of neurodegenerative diseases is well known, its contribution to the genomic dysregulation associated with psychiatric disorders is less well defined. In this study we used the SH-SY5Y cell line and applied RNA-sequencing to explore transcriptomic changes in response to OS before or during neural differentiation. We observed differential expression of many genes, most of which localised to the synapse and were involved in neuronal differentiation. These genes were enriched in schizophrenia-associated signalling pathways, including PI3K/Akt, axon guidance, and signalling by retinoic acid. Interestingly, circulatory system development was affected by both treatments, which is concordant with observations of increased prevalence of cardiovascular disease in patients with NDDs. We also observed a very significant increase in the expression of immunity-related genes, supporting current hypotheses of immune system involvement in psychiatric disorders. While further investigation of this influence in other cell and animal models is warranted, our data suggest that early life exposure to OS has a disruptive influence on neuronal gene expression that may perturb normal differentiation and neurodevelopment, thereby contributing towards overall risk for developing psychiatric diseases.