AKT inhibition interferes with the expression of immune checkpoint proteins and increases NK-induced killing of HL60-AML cells.

OBJECTIVE: To determine the role of the AKT pathway in the regulating of natural Killer-induced apoptosis of acute myeloid leukemia cells and to characterize the associated molecular mechanisms. METHODS: BALB/c nude mice were injected with HL60 cells to induce a xenogenic model of subcutaneous leukemic tumors. Mice were treated with perifosine, and their spleens were analyzed using biometry, histopathology, and immunohistochemistry. Gene expression analysis in leukemia cells was performed by real-time PCR. Protein analysis of leukemia and natural Killer cells was performed by flow cytometry. AKT inhibition in HL60 cells, followed by co-culture with natural Killer cells was performed to assess cytotoxicity. Apoptosis rate was quantified using flow cytometry. RESULTS: Perifosine treatment caused a reduction in leukemic infiltration in the spleens of BALB/c nude mice. In vitro , AKT inhibition reduced HL60 resistance to natural Killer-induced apoptosis. AKT inhibition suppressed the immune checkpoint proteins PD-L1, galectin-9, and CD122 in HL60 cells, but did not change the expression of their co-receptors PD1, Tim3, and CD96 on the natural Killer cell surface. In addition, the death receptors DR4, TNFR1, and FAS were overexpressed by AKT inhibition, thus increasing the susceptibility of HL60 cells to the extrinsic pathway of apoptosis. CONCLUSION: The AKT pathway is involved in resistance to natural Killer-induced apoptosis in HL60 cells by regulating the expression of immune suppressor receptors. These findings highlight the importance of AKT in contributing to immune evasion mechanisms in acute myeloid leukemia and suggests the potential of AKT inhibition as an adjunct to immunotherapy.

In vitro differentiation of myeloid suppressor cells (MDSC-like) from an immature myelomonocytic precursor THP-1.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population with a potent suppressor profile that regulates immune responses. These cells are one of the main components of the microenvironment of several diseases, including solid and hematologic tumors, autoimmunities, and chronic inflammation. However, their wide use in studies is limited due to they comprehend a rare population, which is difficult to isolate, expand, differentiate, and maintain in culture. Additionally, this population has a complex phenotypic and functional characterization. OBJECTIVE: To develop a protocol for the in vitro production of MDSC-like population from the differentiation of the immature myeloid cell line THP-1. METHODS: We stimulated THP-1 with G-CSF (100 ng/mL) and IL-4 (20 ng/mL) for seven days to differentiate into the MDSC-like profile. At the end of the protocol, we characterized these cells phenotypically and functionally by immunophenotyping, gene expression analysis, cytokine release dosage, lymphocyte proliferation, and NK-mediated killing essays. RESULTS: We differentiate THP-1 cells in an MDSC-like population, named THP1-MDSC-like, which presented immunophenotyping and gene expression profiles compatible with that described in the literature. Furthermore, we verified that this phenotypic and functional differentiation did not deviate to a macrophage profile of M1 or M2. These THP1-MDSC-like cells secreted several immunoregulatory cytokines into the microenvironment, consistent with the suppressor profile related to MDSC. In addition, the supernatant of these cells decreased the proliferation of activated lymphocytes and impaired the apoptosis of leukemic cells induced by NK cells. CONCLUSIONS: We developed an effective protocol for MDSC in vitro production from the differentiation of the immature myeloid cell line THP-1 induced by G-CSF and IL-4. Furthermore, we demonstrated that THP1-MDSC-like suppressor cells contribute to the immune escape of AML cells. Potentially, these THP1-MDSC-like cells can be applied on a large-scale platform, thus being able to impact the course of several studies and models such as cancer, immunodeficiencies, autoimmunity, and chronic inflammation.

AKT inhibition interferes with the expression of immune checkpoint proteins and increases NK-induced killing of HL60-AML cells

ABSTRACT Objective To determine the role of the AKT pathway in the regulating of natural Killer-induced apoptosis of acute myeloid leukemia cells and to characterize the associated molecular mechanisms. Methods BALB/c nude mice were injected with HL60 cells to induce a xenogenic model of subcutaneous leukemic tumors. Mice were treated with perifosine, and their spleens were analyzed using biometry, histopathology, and immunohistochemistry. Gene expression analysis in leukemia cells was performed by real-time PCR. Protein analysis of leukemia and natural Killer cells was performed by flow cytometry. AKT inhibition in HL60 cells, followed by co-culture with natural Killer cells was performed to assess cytotoxicity. Apoptosis rate was quantified using flow cytometry. Results Perifosine treatment caused a reduction in leukemic infiltration in the spleens of BALB/c nude mice. In vitro , AKT inhibition reduced HL60 resistance to natural Killer-induced apoptosis. AKT inhibition suppressed the immune checkpoint proteins PD-L1, galectin-9, and CD122 in HL60 cells, but did not change the expression of their co-receptors PD1, Tim3, and CD96 on the natural Killer cell surface. In addition, the death receptors DR4, TNFR1, and FAS were overexpressed by AKT inhibition, thus increasing the susceptibility of HL60 cells to the extrinsic pathway of apoptosis. Conclusion The AKT pathway is involved in resistance to natural Killer-induced apoptosis in HL60 cells by regulating the expression of immune suppressor receptors. These findings highlight the importance of AKT in contributing to immune evasion mechanisms in acute myeloid leukemia and suggests the potential of AKT inhibition as an adjunct to immunotherapy.

The Bone Marrow Microenvironment Mechanisms in Acute Myeloid Leukemia.

Bone marrow (BM) is a highly complex tissue that provides important regulatory signals to orchestrate hematopoiesis. Resident and transient cells occupy and interact with some well characterized niches to produce molecular and cellular mechanisms that interfere with differentiation, migration, survival, and proliferation in this microenvironment. The acute myeloid leukemia (AML), the most common and severe hematological neoplasm in adults, arises and develop in the BM. The osteoblastic, vascular, and reticular niches provide surface co-receptors, soluble factors, cytokines, and chemokines that mediate important functions on hematopoietic cells and leukemic blasts. There are some evidences of how AML modify the architecture and function of these three BM niches, but it has been still unclear how essential those modifications are to maintain AML development. Basic studies and clinical trials have been suggesting that disturbing specific cells and molecules into the BM niches might be able to impair leukemia competencies. Either through niche-specific molecule inhibition alone or in combination with more traditional drugs, the bone marrow microenvironment is currently considered the potential target for new strategies to treat AML patients. This review describes the cellular and molecular constitution of the BM niches under healthy and AML conditions, presenting this anatomical compartment by a new perspective: as a prospective target for current and next generation therapies.

Author Correction: Influence of high glucose on mesangial cell-derived exosome composition, secretion and cell communication.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Treatment with Mesenchymal Stem Cells Improves Renovascular Hypertension and Preserves the Ability of the Contralateral Kidney to Excrete Sodium.

BACKGROUND: Mesenchymal stem cells (MSC) improve renal function and renovascular hypertension in the 2-kidney 1-clip model (2K-1C). While MSC play an immunomodulatory role, induce neoangiogenesis, and reduce fibrosis, they do not correct sodium loss by the contra-lateral kidney. OBJECTIVES: We investigated the tubular function of both stenotic and contralateral kidneys and the effect of MSC treatment by evaluating diuresis, natriuresis, and the expression of the main water and sodium transporters. METHOD: Adult Wistar rats were allocated into four groups: control (CT), CT+MSC, 2K-1C, and 2K-1C+MSC. MSC (2 × 105) were infused through the tail vein 3 and 5 weeks after clipping. Systolic blood pressure (SBP) was monitored weekly by plethysmography. Six weeks after clipping, 24-hour urine and blood samples were collected for biochemical analysis. Gene expression of the Na/H exchanger-3, epithelial sodium channel, Na/K-ATPase, Na/K/2Cl cotransporter, and aquaporins 1 and 2 (AQP1 and AQP2) were analyzed by RT-PCR. Intrarenal distribution of AQP1 and AQP2 was analyzed by immunohistochemistry. RESULTS: In hypertensive 2K-1C animals, MSC prevented additional increases in BP. AQP1, but not AQP2, was suppressed in the contralateral kidney, resulting in significant increase in urinary flow rate and sodium excretion. Gene expressions of sodium transporters were similar in both kidneys, suggesting that the high perfusing pressure in the contralateral kidney was responsible for increased natriuresis. Contralateral hypertensive kidney showed signs of renal deterioration with lower GFR in spite of normal RPF levels. CONCLUSIONS: MSC treatment improved renal function and enhanced the ability of the contralateral kidney to excrete sodium through a tubular independent mechanism contributing to reduce SBP.

Influence of high glucose on mesangial cell-derived exosome composition, secretion and cell communication.

Mesangial cells stimulated with high glucose (HG) exhibit increased intracellular angiotensin II (AngII) synthesis that is correlated with the upregulation of AngII target genes, such as profibrotic cytokines. The intracrine effects of AngII can be mediated by several molecules transferred to other cells via exosomes (Exos), which play a key role in cellular communication under many physiological and pathological conditions. The aim of this study was to investigate the effects of exosomes derived from HG-stimulated human mesangial cells (HG-HMCs) on normal unstimulated HMCs. Exosomes from HMCs (C-Exos) and HG-HMCs (HG-Exos) were obtained from cell culture supernatants. HMCs were incubated with C-Exos or HG-Exos. HG stimulus induced a change in the amount but not the size of Exos. Both C-Exos and HG-Exos contained angiotensinogen and renin, but no angiotensin converting enzyme was detected. Compared with HMCs treated with C-Exos, HMCs treated with HG-Exos presented higher levels of fibronectin, angiotensinogen, renin, AT1 and AT2 receptors, indicating that HG-Exos modified the function of normal HMCs. These results suggest that the intercellular communication through Exos may have pathophysiological implications in the diabetic kidney.

miR-26a modulates HGF and STAT3 effects on the kidney repair process in a glycerol-induced AKI model in rats.

Acute kidney injury is mostly reversible, and hepatocyte growth factor (HGF) has a relevant role in the tissue repair. MicroRNA (miR)-26a is an endogenous modulator of HGF. The role of miR-26a in the kidney repair process was evaluated in Wistar rats submitted to an acute kidney injury model of rhabdomyolysis induced by glycerol (6 mL/kg). Animals were evaluated 3, 12, 48, 96, and 120 hours after glycerol injection. Serum creatinine (SCr) and gene expression of HGF, c-met, signal transducer and activator of transcription 3 (STAT3), and miR-26a were estimated. Also, tubular NK52E cells were transfected with anti-miR26a and stimulated with Fe3+ for 24 hours to mimic the effects of myoglobin in vitro. SCr was highest after 48 hours. After 96 hours, SCr started to decrease, characterizing the recovery phase, with normalization after 120 hours. HGF expression increased during the onset phase (3 hours), with a low relationship with miR-26a. In contrast, in the recovery phase, the increase in miR-26a was coincident with HGF messenger RNA suppression, suggesting that in the recovery phase, miR-26a may have a role in HGF modulation. Fe3+ induced cellular death after 3 hours and proliferation after 24 hours. There was no correlation between miR-26a and STAT3 during the death phase; however, during the proliferation phase, an increase in STAT3 was paralleled with a decrease in miR-26a. miR-26a silencing induced increases in cell viability and the phosphorylated form of STAT3 protein expression in cells receiving Fe3+ . In conclusion, miR-26a may have a key role in modulating HGF levels after its proliferative effects have been triggered.

Renovascular hypertension: Effects of mesenchymal stem cells in the contralateral hypertensive kidney in rats.

Mesenchymal stem cells (MSC) induced neovascularization and improved renal morphology of the stenotic kidney in 2 kidneys-1 clip (2K-1C) model of renovascular hypertension. The present study evaluated the effects of MSC in the contralateral hypertensive kidney. Three weeks after left renal artery occlusion, MSC were injected into the tail vein of the 2K-1C rats. Renal function and morphology were analyzed in both kidneys. Labeled MSC were found in stenotic and contralateral kidneys. Hypertensive 2K-1C animals presented increased circulating levels of Angiotensin II (Ang II) and renin. MSC prevented the progressive increase of blood pressure and reduced circulating Ang II and renin levels. Stenotic kidney showed reduced renal plasma flow (RPF) and glomerular filtration rate (GFR), whereas the contralateral kidney had a tendency (p > 0.5) of reduction in GFR in spite of unchanged RPF. MSC treatment caused an improvement in GFR with no effect of on RPF in the stenotic kidney. Contralateral kidney showed increased diuresis and natriuresis that were even higher in MSC-treated animals, indicating that cell treatment improved the capacity of the contralateral kidney to excrete sodium. Contralateral kidney expressed higher levels of inflammatory cytokines (IL-6, TNF-α) and signs of fibrosis, which were attenuated by MSC treatment. MSC treatment improved the stenotic kidney function, and it was also beneficial to the contralateral hypertensive kidney because it improved the morphology and preserved its capacity to excrete sodium.