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Aerobic exercise training (AET) has emerged as a strategy to reduce cancer mortality, however, the mechanisms explaining AET on tumor development remain unclear. Tumors escape immune detection by generating immunosuppressive microenvironments and impaired T cell function, which is associated with T cell mitochondrial loss. AET improves mitochondrial content and function, thus we tested whether AET would modulate mitochondrial metabolism in tumor-infiltrating lymphocytes (TIL). Balb/c mice were subjected to a treadmill AET protocol prior to CT26 colon carcinoma cells injection and until tumor harvest. Tissue hypoxia, TIL infiltration and effector function, and mitochondrial content, morphology and function were evaluated. AET reduced tumor growth, improved survival, and decreased tumor hypoxia. An increased CD8+ TIL infiltration, IFN-γ and ATP production promoted by AET was correlated with reduced mitochondrial loss in these cells. Collectively, AET decreases tumor growth partially by increasing CD8+ TIL effector function through an improvement in their mitochondrial content and function.

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Regular and moderate exercise is being used for therapeutic purposes in treating several diseases, including cancer, cardiovascular diseases, arthritis, and even chronic kidney diseases (CKDs). Conversely, extenuating physical exercise has long been pointed out as one of the sources of acute kidney injury (AKI) due to its severe impact on the body's physiology. AKI development is associated with increased tubular necrosis, which initiates a cascade of inflammatory responses. The latter involves cytokine production, immune cell (macrophages, lymphocytes, and neutrophils, among others) activation, and increased oxidative stress. AKI can induce prolonged fibrosis stimulation, leading to CKD development. The need for therapeutic alternative treatments for AKI is still a relevant issue. In this context arises the question as to whether moderate, not extenuating, exercise could, on some level, prevent AKI. Several studies have shown that moderate exercise can help reduce tissue damage and increase the functional recovery of the kidneys after an acute injury. In particular, the immune system can be modulated by exercise, leading to a better recovery from different pathologies. In this review, we aimed to explore the role of exercise not as a trigger of AKI, but as a modulator of the inflammatory/immune system in the prevention or recovery from AKI in different scenarios. In AKI induced by ischemia and reperfusion, sepsis, diabetes, antibiotics, or chemotherapy, regular and/or moderate exercise could modulate the immune system toward a more regulatory immune response, presenting, in general, an anti-inflammatory profile. Exercise was shown to diminish oxidative stress, inflammatory markers (caspase-3, lactate dehydrogenase, and nitric oxide), inflammatory cytokines (interleukin (IL)-1b, IL-6, IL-8, and tumor necrosis factor-α (TNF-α)), modulate lymphocytes to an immune suppressive phenotype, and decrease tumor necrosis factor-ß (TGF-ß), a cytokine associated with fibrosis development. Thus, it creates an AKI recovery environment with less tissue damage, hypoxia, apoptosis, or fibrosis. In conclusion, the practice of regular moderate physical exercise has an impact on the immune system, favoring a regulatory and anti-inflammatory profile that prevents the occurrence of AKI and/or assists in the recovery from AKI. Moderate exercise should be considered for patients with AKI as a complementary therapy.

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Background: Conventional thoracotomy (CT) often leads to systemic inflammatory response syndrome (SIRS), which induces several clinical complications. CT remains widely used in low-income institutions. Although minimally invasive surgical procedures, such as robotic surgery (RS), have been used to prevent many of the complications inherit from the surgical procedure. Here, we investigated the protective effect of vagus nerve stimulation (VNS) in a pre-clinical model during CT or RS and postoperative period (POP) relative to clinical complications and inflammatory control. The objective was to compare hemodynamic features and cytokine levels in the blood, lung, and bronchoalveolar lavage (BAL) fluids of animals subjected to CT or RS with or without VNS. Methods: Twenty-four minipigs were subjected to 12 animals CT and 12 animals RS, with or without VNS, and accompanied 24 h later by pulmonary lobectomy. Blood samples for evaluating the hemodynamic parameters were collected before the surgical preparation, immediately after the beginning of VNS, and every 4 h until 24 h after the lobectomy. BAL fluid and lung tissue were collected at the end of the experiment. Cytokine levels were evaluated in the blood, BAL fluid, and lung tissues. Results: VNS maintained a more stable heart rate during POP and decreased the incidence of overall cardiac complications while preventing increase in IL-6 levels 12 h after lobectomy, compared to sham animals. No differences were found in cytokine expression in the BAL fluid and lung tissue in any of the studied groups. Conclusions: Taken together, our data suggested that VNS should be considered a non-pharmacological tool in the prevention of the exacerbated inflammatory response responsible for severe clinical complications, especially in more aggressive surgical procedures.

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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.

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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.

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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.

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OBJECTIVES: Gene fusions are becoming more evident in cancer scenario for either being the driver alterations, or for the great therapeutic target potential in many cases. Our aim was to characterize the BRD4-NOTCH3 fusion correlating with clinical features, and to determine the frequency of this fusion in the oncological population. MATERIAL AND METHODS: One patient diagnosed with lung adenocarcinoma at Hospital Sírio-Libanês (Brazil) was included. Foundation Medicine database was searched for all BRD4-NOTCH3 fusions among 233,804 specimens. RESULTS: A 76-year-old male patient was diagnosed with lung adenocarcinoma. Molecular assessments demonstrated negative ALK and EGFR, with PD-L1 expression positive by 60 %. He was treated with first line chemotherapy and second line immunotherapy. Subsequent treatments resume re-exposures to chemotherapy with poor responses. A next-generation sequencing (NGS) based assay was performed in the tumor biopsy, revealing mainly mutation in STK11, microsatellite stability, TMB-intermediate, MYC amplification and a BRD4-NOTCH3 fusion. The breakpoint analysis of this fusion indicates that BRD4 active domains are preserved, suggesting that it maintained DNA binding activity, as well as its capacity to be halt by BET inhibitors. Foundation Medicine database was searched for all BRD4-NOTCH3 fusions among more than 230-thousand specimens and it was found in 87 new cases in a rate of 0.04 % occurrence in solid tumors, predominately in gynecological cancers. The same rate was found when we analyzed a different dataset. CONCLUSION: In conclusion, this is the first report of the BRD4-NOTCH3 gene fusion associated with clinical characterization and, although rare, the occurrence of this fusion is constant in different population. Our data suggest that this fusion has great potential to targeted-therapy.

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Cartilage lesions and osteoarthritis (OA) presents an ever-increasing clinical and socioeconomic burden. Synovial inflammation and articular inflammatory environment are the key factor for chondrocytes apoptosis and hypertrophy, ectopic bone formation and OA progression. To effectively treat OA, it is critical to develop a drug that skews inflammation toward a pro-chondrogenic microenvironment. In this narrative and critical review, we aim to see the potential use of immune cells modulation or cell therapy as therapeutic alternatives to OA patients. Macrophages are immune cells that are present in synovial lining, with different roles depending on their subtypes. These cells can polarize to pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes, being the latter associated with wound-healing by the production of ARG-1 and pro-chondrogenic cytokines, such as IL-10, IL-1RA, and TGF-b. Emerging evidence reveals that macrophage shift can be determined by several stimuli, apart from the conventional in vitro IL-4, IL-13, and IL-10. Evidences show the potential of physical exercise to induce type 2 response, favoring M2 polarization. Moreover, macrophages in contact with oxLDL have effect on the production of anabolic mediators as TGF-b. In the same direction, type II collagen, that plays a critical role in development and maturation process of chondrocytes, can also induce M2 macrophages, increasing TGF-b. The mTOR pathway activation in macrophages was shown to be able to polarize macrophages in vitro, though further studies are required. The possibility to use mesenchymal stem cells (MSCs) in cartilage restoration have a more concrete literature, besides, MSCs also have the capability to induce M2 macrophages. In the other direction, M1 polarized macrophages inhibit the proliferation and viability of MSCs and impair their ability to immunosuppress the environment, preventing cartilage repair. Therefore, even though MSCs therapeutic researches advances, other sources of M2 polarization are attractive issues, and further studies will contribute to the possibility to manipulate this polarization and to use it as a therapeutic approach in OA patients.

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Mesenchymal stem cells (MSCs) have been studied as a promising type of stem cell for use in cell therapies because of their ability to regulate the immune response. Although they are classically isolated from the bone marrow, many studies have sought to isolate MSCs from noninvasive sources. The objective of this study was to evaluate how MSCs isolated from the dental pulp of human exfoliated deciduous teeth (SHED) and fragments of the orbicularis oris muscle (OOMDSCs) behave when treated with an inflammatory IFN-γ stimulus, specifically regarding their proliferative, osteogenic, and immunomodulatory potentials. The results demonstrated that the proliferation of SHED and OOMDSCs was inhibited by the addition of IFN-γ to their culture medium and that treatment with IFN-γ at higher concentrations resulted in a greater inhibition of the proliferation of these cells than treatment with IFN-γ at lower concentrations. SHED and OOMDSCs maintained their osteogenic differentiation potential after stimulation with IFN-γ. Additionally, SHED and OOMDSCs have been shown to have low immunogenicity because they lack expression of HLA-DR and costimulatory molecules such as CD40, CD80, and CD86 before and after IFN-γ treatment. Last, SHED and OOMDSCs expressed the immunoregulatory molecule HLA-G, and the expression of this antigen increased after IFN-γ treatment. In particular, an increase in intracellular HLA-G expression was observed. The results obtained suggest that SHED and OOMDSCs lack immunogenicity and have immunomodulatory properties that are enhanced when they undergo inflammatory stimulation with IFN-γ, which opens new perspectives for the therapeutic use of these cells.