Rac GTPases in acute myeloid leukemia cells: Expression profile and biological effects of pharmacological inhibition.

Acute myeloid leukemia (AML) is a highly heterogeneous hematological neoplasm with low survival rates. Thus, the investigation of new therapeutic targets is essential. The Rac subfamily of GTPase proteins has been shown to participate in the physiopathology of hematological malignancies. However, their expression and function in AML remain unclear. In this study, we evaluated Rac1, Rac2 and Rac3 gene expressions in AML and their impact on clinical outcomes. We further investigated the effects of the in vitro treatment with a Rac inhibitor (EHT-1864) on AML cell lines. Rac3 expression was increased in AML derived from myelodysplastic syndromes compared to healthy donors. Rac2 expression did not differ between AML patients and healthy donors, but de novo AML patients with higher Rac2 presented lower overall survival. Oncogenic pathway gene-sets related to AKT/mTOR were identified as associated with Rac1, Rac2 and Rac3 expressions. EHT-1864 treatment reduced the viability of OCI-AML3, KG1 and Kasumi-1 cells in a time and dose-dependent manner. In OCI-AML3 cells, treatment with EHT-1864 induced apoptosis, autophagy, and led to the accumulation of cells in the G1 phase of the cell cycle. These changes were concomitant with alterations in p53 and cyclins. Dowregulation of the PI3K/AKT/mTOR pathway was also observed. Interestingly, the combined treatment of EHT-1864 and low doses of daunorubicin enhanced OCI-AML3 cell apoptosis. In conclusion, Rac2 expression is a prognostic factor in AML and our preclinical results suggest that Rac inhibition may be an attractive mechanism to compose the antineoplastic strategy for this disease.

Protective effect of green tea and epigallocatechin-3-gallate in a LPS-induced systemic inflammation model.

Inflammation causes severe dysregulation of organ functions, via the development of oxidative stress and inflammation damage. Polyphenol compounds found in green tea (GTE), including the most important component epigallocatechin-3-gallate (EGCG), have a great therapeutic potential. Here, protective properties of GTE and EGCG against lipopolysaccharide (LPS)-induced inflammation are explored. To this end, the effects of GTE and EGCG were studied on LPS challenged macrophages. Mice received GTE (250 mg/kg/d/p.o) or EGCG (25 mg/kg/d/i.p.) for 7 d, before the inflammation shock was provoked with a single intraperitoneal injection of LPS. The frequencies of lymphocytes CD4+, CD8+, NK1-1+ and CD4+CD25highFOXP3+ (Treg), macrophages CD11b+F480+, monocytes CD11b+Ly6Clow/high, neutrophils CD11b+Ly6G+, MDSCs CD11b+Gr-1high, M2/N2-like phenotype CD206+ and M1-like phenotype CD86+ in spleen, bone marrow and peripheral blood were determined. In vitro studies revealed that GTE and EGCG significantly attenuated LPS-induced CD80 expression and increased the CD163 expression, showing a potential to reduce the macrophage inflammatory phenotype. In vivo, GTE and EGCG inhibited the inflammation, mainly by reducing M1-macrophages and increasing Treg cells in the bone marrow. In addition, GTE and EGCG increase M2-macrophages, N2-neutrophils and Tregs in the spleen and blood and block the migration of monocytes from the bone marrow to the peripheral blood. These findings indicate that EGCG and GTE prevent LPS-induced inflammatory damage contributing to restoring the immune system homeostasis.

Artesunate strongly modulates myeloid and regulatory T cells to prevent LPS-induced systemic inflammation.

Lipopolysaccharide (LPS) is the major component of the outer membrane of Gram-negative bacteria and is usually administrated to establish models of inflammation. Artesunate (ART), a water-soluble artemisinin derivative, displays multiple pharmacological actions against tumors, viral infections, and inflammation, and has been used as a therapeutic weapon against malaria. In this study, our aim was to evaluate whether ART pretreatment is capable of preventing inflammation induced by LPS. BALB/c mice were treated with 100 mg/kg of ART i.p. for 7 days followed by a single dose of LPS. ART pretreatment led to an improvement in clinical score, prevented alterations in biochemical markers, and reestablished the platelet counts. Flow cytometry analysis showed that ART protected the inflammation mainly by reducing the percentage of M1 macrophages while increasing M2 macrophages and a reestablishment of classical monocytes in the BM. In the spleen, ART pretreatment increased N2 neutrophils, myeloid-derived suppressor cells (MDSC), and regulatory T cells, the latter was also increased in peripheral blood. In addition, a marked decrease in inflammatory cytokines and chemokines was observed in the ART treated group. Our data suggest that ART prevents inflammation, reducing tissue damage and restoring homeostasis.

Artesunate Switches Monocytes to an Inflammatory Phenotype with the Ability to Kill Leukemic Cells.

Monocytes are components of the tumor microenvironment related to cancer progression and immune escape. Therapeutic strategies for reprogramming monocytes from a tumor-supporting phenotype towards a tumoricidal phenotype are of great interest. Artesunate (ART) may be an interesting option for cancer treatment; however, the role of ART in regulating the inflammatory tumor microenvironment has not yet been investigated. Our aim is to evaluate the immunomodulatory potential of ART in vitro in human primary monocytes. ART treatment induced an increase in inflammatory monocytes (CD14highCD16-) with HLA-DR high expression and MCP-1/IL-1ß release. On the other hand, ART treatment reduced CD206 and CD163 expression, and abolished the monocyte population known as non-classical and intermediate. Leukemia cells in contact with monocytes programmed with ART presented enhanced in vitro apoptosis suggesting that monocytes acquired the ability to kill leukemic cells. ART induced changes in the monocyte phenotype were mediated by JAK2/STAT3 downregulation. The induction of immunosuppressive environment is an important step for cancer progression. ART showed an immunomodulatory activity, leading immune cells to an antitumor phenotype and could be a candidate for immunotherapy in cancer patients.

Artemisinins induce endoplasmic reticulum stress in acute leukaemia cells in vitro and in vivo.

Loss of endoplasmic reticulum (ER) homeostasis leads to ER stress, thus prolonged activation can lead to apoptosis. Herein, artesunate (ART) induced ER stress in leukaemia cells, resulting in eIF2α phosphorylation, activation of transcription factor 4, subsequent CHOP upregulation and XBP1 splicing. Furthermore, in vitro cyclin/CDKs reduction induced G1-phase arrest. An in vivo xenograft model showed a consistent pattern of ART in reducing tumour burden, supporting roles in the UPR pathway, which we speculate could lead to apoptosis by NOXA activation. Moreover, ART were capable of increasing the survival of mice. Taken together, our data indicate that ART may represent an interesting weapon to fight leukaemia.

Impaired expression of CXCL5 and matrix metalloproteinases in the lungs of mice with high susceptibility to Streptococcus pneumoniae infection

Introduction: Streptococcus pneumoniae colonizes the nasopharynx of healthy individuals establishing a commensal relationship with the host. In some conditions, bacteria invade the lower respiratory tract and innate immune responses are crucial to avoid diseases such as pneumonia, sepsis, or meningitis. Methods: Here, we compared the susceptibility to pneumococcal respiratory infection of two outbred mouse lines, AIRmin and AIRmax, selected for low or high acute inflammatory responses, respectively. Results: AIRmin mice showed increased susceptibility to infection with different pneumococcal serotypes, when compared to AIRmax. Significant higher numbers of alveolar macrophages expressing the CD206 mannose receptor were observed in AIRmin mice when compared to AIRmax mice. Despite this difference, secretion of several cytokines and chemokines in the respiratory tract of AIRmin and AIRmax mice, after infection, was similar. The only exception was CXCL5, which was highly induced after pneumococcal infection in AIRmax mice but not in AIRmin mice. Reduced expression of the matrix metalloproteinases (MMP) 2, 3, 8, and 9, as well as reduced activities of MMPs were also observed in the lungs of AIRmin mice, after infection. Such impaired responses may have contributed to the low influx of neutrophils observed in the airways of these mice. Finally, high percentages of macrophages and neutrophils in apoptosis or necrosis, at the site of infection, were also observed in AIRmin mice, suggesting that leukocyte functionality is also compromised. Conclusions: Our results indicate that CXCL5 and MMPs contribute to the resistance to pneumococcal infection in mice.

Impaired expression of CXCL5 and matrix metalloproteinases in the lungs of mice with high susceptibility to Streptococcus pneumoniae infection

Introduction: Streptococcus pneumoniae colonizes the nasopharynx of healthy individuals establishing a commensal relationship with the host. In some conditions, bacteria invade the lower respiratory tract and innate immune responses are crucial to avoid diseases such as pneumonia, sepsis, or meningitis. Methods: Here, we compared the susceptibility to pneumococcal respiratory infection of two outbred mouse lines, AIRmin and AIRmax, selected for low or high acute inflammatory responses, respectively. Results: AIRmin mice showed increased susceptibility to infection with different pneumococcal serotypes, when compared to AIRmax. Significant higher numbers of alveolar macrophages expressing the CD206 mannose receptor were observed in AIRmin mice when compared to AIRmax mice. Despite this difference, secretion of several cytokines and chemokines in the respiratory tract of AIRmin and AIRmax mice, after infection, was similar. The only exception was CXCL5, which was highly induced after pneumococcal infection in AIRmax mice but not in AIRmin mice. Reduced expression of the matrix metalloproteinases (MMP) 2, 3, 8, and 9, as well as reduced activities of MMPs were also observed in the lungs of AIRmin mice, after infection. Such impaired responses may have contributed to the low influx of neutrophils observed in the airways of these mice. Finally, high percentages of macrophages and neutrophils in apoptosis or necrosis, at the site of infection, were also observed in AIRmin mice, suggesting that leukocyte functionality is also compromised. Conclusions: Our results indicate that CXCL5 and MMPs contribute to the resistance to pneumococcal infection in mice.