Fcγ receptors on aging neutrophils

Objective: Neutrophils are key effector cells of the innate immune system. They recognize antigens through membrane receptors, which are expressed during their maturation and activation. Neutrophils express FcγRII (CD32), FcγRIII (CD16), and FcγRI (CD64) after being activated by different factors such as cytokines and bacterial products. These receptors are involved with phagocytosis of IgG-opsonized microbes and enhance defense mechanisms. Based on that, our study seeks to compare the expression of FcγRII, FcγRIII, FcγRI, and CD11b on neutrophils from elderly and young subjects and their expression after in vitro activation with cytokines and LPS. Methodology: Neutrophils were isolated from human peripheral blood and from mice bone marrow by density gradient. After isolation, FCγRs expression was immediately analyzed by flow cytometry or after in vitro stimulation. Results: In freshly isolated cells, the percentage of FcγRIIIb+ and CD11b+ neutrophils were higher in samples from young individuals; FcγRIIIa expression was more prominent on aged neutrophils; FcγRIA expression was similar in all samples analyzed. Exposure to CXCL8 and LPS resulted in a higher percentage of FcγRIa+ neutrophils on elderly individuals’ samples but lower when compared with neutrophils from young donors. We observed that LPS caused an increase in FcγRIIa expression on aging human neutrophils. In contrast, FcγRIIIb expression in response to CXCL8 and LPS stimulation was not altered in the four groups. CD11b expression was lower in neutrophils from elderly individuals even in response to LPS and CXCL8. In mice, we observed differences only regarding CD11b expression, which was increased on aged neutrophils. LPS exposure caused an increase in all FcγRs. Conclusions: Our results suggest that, in humans, the overall pattern of FcγR expression and integrin CD11b are altered during aging and immunosenescence might contribute to age-related infection

Caracterização da relação funcional entre macrófagos, fenótipo célula-tronco de câncer e fenômeno de transição epitélio-mesenquimal no carcinoma epidermóide de boca

Oral squamous cell carcinoma (OSCC) is one of the most common malignant neoplasms of the head and neck, and the major prognostic factor is the presence of metastatic lesions in cervical lymph nodes. Studies have shown that cancer stem cells (CSCs) might be involved with migration and invasion process through the epithelial-to-mesenchymal transition (EMT) in tumor cells. It is known that cellular and soluble components of tumor microenvironment influence the EMT process, and macrophages represent the major class of cells that are recruited to the tumor site in different cancers. Furthermore, TGF- signaling has been described as a critical regulator of EMT in cancer cells and can sustain CSCs properties. In view of that, the purpose of this study was to determine the functional relationship between CSC, EMT, and macrophages in OSCC. In paper 1, we analyzed the in vitro and in vivo behavior of two CSCs subpopulations, CD44HighESALow and CD44HighESAHigh sorted from the OSCC cell line, LUC4. Our findings suggested that the subpopulation CD44HighESAHigh is the one that carries the most stemness features. However, what induced or sustained that phenotype remained to be clarified. In paper 2, the influence of macrophages on CSCs was analyzed by directly co-culturing the subpopulations of CSC with macrophages or culturing CSC with conditioned medium from macrophages. Our results suggested that macrophages promote EMT in CSC, and that influence was probably mediated by TGF- signaling. Altogether, our results showed that tumor heterogeneity must be considered and deeply characterized to better determine the therapeutic approach.O carcinoma epidermóide de boca (CEB) é uma das neoplasias malignas mais comuns da região da cabeça e do pescoço e o fator prognóstico com impacto mais significante nessa doença é a presença de metástase em linfonodos cervicais. Evidências mostram que células-tronco de câncer (CSCs, do inglês cancer stem cells) podem estar envolvidas nos processos de migração e invasão por meio da ativação do fenômeno de transição epitélio-mesenquimal (TEM) nas células tumorais. Sabe-se, que o processo de TEM pode ser influenciado por componentes celulares e solúveis presentes no microambiente tumoral e que, dentre as células infiltrantes, os macrófagos representam a principal população leucocitária recrutada, sendo detectado em maior número em diferentes neoplasias. Ademais, a sinalização mediada por TGF- tem sido descrita como essencial para regulação de TEM em células tumorais bem como para a manutenção das propriedades, diferenciação e função das CSCs. Nesse contexto, a proposta deste estudo foi determinar a relação funcional entre CSC, TEM e macrófagos em CEB. No artigo 1, analisamos o comportamento in vivo e in vitro de duas subpopulações de CSC, CD44HighESAHigh e CD44HighESALow, presentes na linhagem de CEB LUC4. Nossos achados sugeriram que a subpopulação CD44HigESAHigh é a subpopulação que mais carrega características de células-tronco. Entretanto, o que induz ou sustenta esse fenótipo ainda não foi esclarecido. No artigo 2, a influência dos macrófagos nas CSC foi analisada através da co-cultura direta das subpopulações de CSC com macrófagos ou por meio da cultura das CSC com médio condicionado obtido da cultura de macrófagos. Nossos resultados demonstraram que os macrófagos promovem TEM nas CSC e que essa influência é mediada pela sinalização de TGF-. Juntos, nossos resultados demonstraram a importância de se considerar a heterogeneidade tumoral para melhor traçar estratégias terapêuticas

Role of ST2 receptor in squamous cell carcinoma development

O carcinoma espinocelular (CEC) é um dos cânceres humanos mais incidentes. A despeito do entendimento da fisiopatologia do CEC, as opções terapêuticas ainda são limitadas e o(s) exato(s) mecanismo(s) envolvido(s) na progressão deste tipo de tumor ainda não foi descrito. Estudos recentes mostram a existência de uma associação direta entre a resposta imune TH1 e um melhor prognóstico em pacientes com CEC. Aumento da expressão de componentes do eixo IL-33/ST2 foi demonstrado contribuir para transformação neoplásica em diversos modelos tumorais, incluindo cânceres de estômago e de mama. Trabalho recente do nosso e de outros laboratórios indicam que IL-33 pode impedir a resposta imune TH1 . Baseado nessas observações, a hipótese testada foi que o impedimento da resposta imune pela interação IL-33/ST2 pode contribuir para iniciação e progressão do CEC. Utilizando modelo de carcinogênese química em camundongos WT e deficientes de ST2 (ST2KO), os resultados mostram que a deficiência de ST2 leva a uma notável redução da severidade das lesões 20 semanas após a carcinogênese química, sugerindo que a sinalização ST2 é necessária para o desenvolvimento tumoral neste modelo. Análises do infiltrado inflamatório presente nas lesões em camundongos WT e ST2KO revelaram redução significativa nas percentagens de macrófagos, células T CD4+ e células dendríticas, mas não em células T CD8+, células B e células natural killer (NK) no microambiente tumoral de camundongos ST2KO. Além disso, células NK esplênicas isoladas de camundongos ST2KO exibiram atividade citotóxica aumentada contra células YAC quando comparado com células de camundongos do grupo controle (WT). Os resultados indicam que a via IL-33/ST2 contribui para o desenvolvimento de carcinoma espinocelular recrutando células T CD4+, macrófagos e células dendríticas e reduzindo a citotoxicidade de células NK.Squamous cell carcinoma (SCC) is the second most common form of skin cancer and is most commonly observed in photo-exposed areas of the body. The mechanism(s) involved in the progression of this tumor are unknown. Recent studies have shown that there is a direct association between a TH1-related immune response and a better prognosis in patients with SCC. Increased expression of the IL33/ST2 axis components has been demonstrated to contribute to neoplastic transformation in several tumor models, including gastric and breast cancer. Recent work from ours and other laboratories indicate that can IL-33 impair TH1-type immune responses. Based on these observations, we hypothesized that TH1-type immune response impairment by IL33/ST2 could contribute to the initiation and progress SCC. We found that ST2 deficiency led to a marked decreased in severity of skin lesions at 20 weeks post-DMBA, suggesting that ST2 signaling is necessary for tumor development in this model. Analysis of tumor lesions in WT and ST2KO mice revealed that lack of ST2 led to a specific and significant reduction in the frequency of macrophages, T CD4+ and dendritic cells, but not CD8+, B and NK cells. In addition, splenic NK cells isolated from DMBA-treated ST2KO mice exhibited increased cytotoxicity activity against YAC cells targets when compared with WT splenic NK cells in the same cytotoxic assay. Altogether, our findings indicate that IL-33/ST2 pathway contributes to the SCC development by recruitment T CD4+ cells, macrophages, and dendritic cells and impairing NK cytotoxicity

Effects of budlein A on human neutrophils and lymphocytes

Sesquiterpene lactones (SLs) are the active constituents of a variety of medicinal plants used in traditional medicine for the treatment of inflammatory diseases and other ailments. Objective In this study, we evaluated whether budlein A modulates the activation of innate and adaptive immune cells such as neutrophils and lymphocytes. Material and Methods Our research group has investigated several plant species and several compounds have been isolated, identified, and their medical potential evaluated. Budlein A is a SL isolated from the species Aldama buddlejiformis and A. robusta (Asteraceae) and shows anti-inflammatory and anti-nociceptive activities. Advances in understanding how plant-derived substances modulate the activation of innate and adaptive immune cells have led to the development of new therapies for human diseases. Results Budlein A inhibited MPO activity, IL-6, CXCL8, IL-10, and IL-12 production and induces neutrophil apoptosis. In contrast, budlein A inhibited lymphocyte proliferation and IL-2, IL-10, TGF-β, and IFN-γ production, but it did not lead to cell death. Conclusions Collectively, our results indicate that budlein A shows distinct immunomodulatory effects on immune cells

Effects of budlein A on human neutrophils and lymphocytes

ABSTRACT Sesquiterpene lactones (SLs) are the active constituents of a variety of medicinal plants used in traditional medicine for the treatment of inflammatory diseases and other ailments. Objective In this study, we evaluated whether budlein A modulates the activation of innate and adaptive immune cells such as neutrophils and lymphocytes. Material and Methods Our research group has investigated several plant species and several compounds have been isolated, identified, and their medical potential evaluated. Budlein A is a SL isolated from the species Aldama buddlejiformis and A. robusta (Asteraceae) and shows anti-inflammatory and anti-nociceptive activities. Advances in understanding how plant-derived substances modulate the activation of innate and adaptive immune cells have led to the development of new therapies for human diseases. Results Budlein A inhibited MPO activity, IL-6, CXCL8, IL-10, and IL-12 production and induces neutrophil apoptosis. In contrast, budlein A inhibited lymphocyte proliferation and IL-2, IL-10, TGF-β, and IFN-γ production, but it did not lead to cell death. Conclusions Collectively, our results indicate that budlein A shows distinct immunomodulatory effects on immune cells

Ganoderma lucidum polysaccharides associated with 5-Fluorouracil impair OSCC tumorigenesis in vitro

Backgrounds: Ganoderma lucidum polysaccharides have been shown several anti-cancer, anti-inflammatory, and immunomodulatory properties among studies with different tumor models and its use with advanced and conventional combination therapies is a world trend. The administration of 5-Fluorouracil is already used as chemotherapy for many tumors and works on tumor remission; however, its adverse effects are still severe, impoverishing treatment and quality of life for patients. Cancer stem cells represent a subpopulation of cells with defense mechanisms against chemotherapy agents and are the main cause of relapses and metastases in cancer treatments. Also, the Epithelial-Mesenchymal Transition program increases properties related to tumor malignancy and mortality. In this scenario, the aim of the study is to evaluate the effects of Ganoderma lucidum polysaccharides in combination with 5-Fluorouracil on the subpopulation of cancer stem cells present in the human oral squamous carcinoma cell line SCC-9. Methods: SCC-9 cells were treated in vitro for 72 h with different 5-Fluorouracil low doses, associated or not with Ganoderma lucidum polysaccharides. Cells maintained with culture media or cisplatin were used as control. All the cells were evaluated for cytotoxicity, cancer stem cells, and Epithelial-Mesenchymal Transition properties. Results: The associated treatment avoided proliferation, delayed migration, slightly modified morphology of cells, increased apoptosis, decreased colony and blocked spheres formation, and downregulated cancer stem cells, Epithelial-Mesenchymal Transition, and ABC drug transporters expression. In addition, Ganoderma lucidum polysaccharides + 5-Fluorouracil changed the treated cells into a non-cancer stem cell phenotype, a characteristically not resistant and less proliferative population. The 5-Fluorouracil treatment alone showed remarkable modification in cellular morphology, apoptosis, and absence of holoclones; however, it upregulated the molecular expression of cancer stem cells' hallmarks. Conclusions: These findings demonstrate that combining Ganoderma lucidum polysaccharides with a low dose of 5-Fluorouracil is effective against oral squamous cell carcinoma in vitro by enhancing the cells' sensitivity to drugs and reducing the characteristics associated with cancer stem cells (CSCs). This suggests the possibility of reducing conventional chemotherapy doses and improving oral squamous cell carcinoma treatment. It also highlights the potential for this combination to be used as an adjunct in Complementary Alternative Medicine (CAM)