Reprogramação metabólica e de função do macrófago por crotoxina

Macrophages (MØ) are cells with high plasticity that can be reprogrammed into two distinct phenotypes – MØ M1 (pro-inflammatory) and MØ M2 (pro-resolving) – according to the nature of the disorder detected in the organism. Experimentally, it is already possible to induce the reprogramming of macrophages to M1 and M2 and the functional and metabolic status of both phenotypes have already been characterized. Previous studies by our group have shown that Crotoxin (CTX) exerts two actions on macrophages that make them suitable in cell therapy: 1) it alters their metabolic and functional profile and 2) these effects are long-lasting. Such effects are observed after a single, short exposure to the toxin. In this context, this research sought to characterize the metabolism and functional status of MØ treated with different concentrations of CTX, with the aim of verifying whether in each concentration it is possible to identify different phenotypes and activation states of the already identified M1 and M2 profiles, in order to seek to reprogram these cells according to specific pathophysiological processes. The capacity for phenotypic reprogramming of monocytes induced by CTX was evaluated in in vitro assays, using cells of the human monocytic lineage (THP-1), previously incubated with RPMI medium (control), or LPS (1ug/mL) or tumor conditioned medium of MCF7 cells (MCT) for 48 hours. Subsequently, they were incubated in the absence (control) or presence of CTX at different concentrations (2 nM; 10 nM; 50 nM and 100 nM) for 1 hour. After the different incubation protocols with the toxin, the differentiation and phenotype markers were analyzed using the qPCR technique, and the latter were also analyzed using flow cytometry. To evaluate the effects of treatments on cellular functions, the phagocytosis capacity, the generation of reactive oxygen species (H2O2) and the production of cytokines by MØ were observed. Cellular metabolism was evaluated through gene expression and activity of key enzymes of the glycolytic pathway. The data presented show that THP-1 cells are satisfactorily differentiated by LPS (1 µg/mL) and tumor environment-MCT (vol. obtained from 1x106 /mL) and these cells have their functions modulated distinctly by CTX, depending on the concentration used. CTX had a dual action on the functions of these human macrophages, since it induced phagocytosis activity, while at the same time stimulated the production of H2O2 in control cells. However, these effects vary depending on the microenvironment stimuli, since, in a tumor and inflammatory context, phagocytosis was preserved in a concentration-dependent manner, concomitant with the increase in H2O2 release in some concentrations. Regarding the metabolic pathways, the undifferentiated THP-1 cells did not show alteration in the gene expression of any of the evaluated glycolytic enzymes, under the effect of any concentration of CTX, suggesting that the toxin does not modulate the expression of these enzymes in the THP-1 cell in monocyte condition. However, when stimulated by LPS and MCT, CTX modulated the gene expression of some enzymes: the 50 nM concentration increased the expression of enzymes from metabolic pathways that are more active in M1 (PFK, FAS and citrate synthase) and also reduced the activity of the enzyme CPT-1 (lipolysis), wich hight activity is related to M2. These effects were accompanied by an increase in pro-inflammatory cytokines. In the inflammatory environment (LPS) the concentration of 10 nM of the toxin promoted a concomitant positive marking of both M1 and M2 markers. Also, at 100 nM CTX promoted parallel release of pro- and anti-inflammatory cytokines. Taken together, the results allow us to state that CTX modulates functions and bioenergetic pathways of human macrophages, depending on the concentration used and the microenvironment stimulus involved.

Os macrófagos (MØ) são células com alta plasticidade que podem ser reprogramados em dois fenótipos distintos – MØ M1 (pró-inflamatórios) e MØ M2 (pró-resolutivos) – de acordo com a natureza do distúrbio detectado no organismo. Experimentalmente, já é possível induzir a reprogramação de macrófagos a M1 e M2 e o estado funcional e metabólico de ambos os fenótipos já foram caracterizados. Estudos prévios de nosso grupo demostraram que a Crotoxina (CTX) exerce em macrófagos duas ações que os tornam aplicáveis em terapia celular: 1) altera seu perfil metabólico e funcional e 2) estes efeitos são de longa duração. Tais efeitos são observados após uma única e curta exposição à toxina. Neste contexto, esta pesquisa buscou caracterizar o metabolismo e o estado funcional de MØ tratados com diferentes concentrações de CTX, com o objetivo de verificar se em cada concentração é possível identificar fenótipos distintos e intermediários aos perfis M1 e M2 já descritos, para assim buscar reprogramar essas células de acordo com processos fisiopatológicos específicos. A capacidade de reprogramação fenotípica de monócitos induzida pela CTX foi avaliada em ensaios in vitro, utilizando células da linhagem monocítica humana (THP-1), incubadas previamente com meio RPMI (controle), ou LPS (1ug/mL) ou meio condicionado tumoral de MCF-7 (MCT) por 48 horas. Posteriormente, elas foram incubadas na ausência (controle) ou presença de CTX em diferentes concentrações (2 nM; 10 nM; 50 nM e 100 nM) ao longo de 1 hora. Após os diferentes protocolos de incubação com a toxina foram analisados os marcadores de diferenciação e de fenótipos, por meio da técnica de qPCR, sendo que os últimos também foram analisados por meio de citometria de fluxo. Para avaliar os efeitos dos tratamentos sobre as funções celulares, foram analisadas a capacidade de fagocitose, a geração de espécies reativas de oxigênio (H2O2) e a produção de citocinas pelos MØ. O metabolismo celular foi avaliado por meio da expressão gênica e atividade das enzimas chave da via glicolítica. Os dados apresentados evidenciam que células THP-1 são satisfatoriamente diferenciadas por LPS (1 µg/mL) e meio condicionado tumoral-MCT (Vol. obtido de 1x106 /mL) e essas células têm suas funções moduladas distintamente pela CTX, a depender da concentração utilizada. A CTX acarretou ação dual sobre as funções destes macrófagos humanos, uma vez que induziu inibição da atividade de fagocitose, ao mesmo tempo que estimulou a produção de H2O2 em células controle. Porém, esses efeitos mudam dependendo dos estímulos do microambiente, já que, em contexto tumoral e inflamatório a fagocitose foi preservada de modo concentração-dependente, acompanhada do aumento da liberação de H2O2 em algumas concentrações. Em relação às vias metabólicas, as células THP-1 não diferenciadas não apresentaram alteração na expressão gênica de nenhuma das enzimas glicolíticas avaliadas, sob o efeito de quaisquer concentrações da CTX, sugerindo que a toxina não modula a expressão dessas enzimas na célula THP-1 na condição de monócito. Porém, quando estimuladas por LPS e MCT a CTX modulou a expressão gênica de algumas enzimas: a concentração de 50 nM aumentou a expressão de enzimas de vias metabólicas que estão mais ativas em M1 (PFK, FAS e citrato sintase) e ainda reduziu a atividade da enzima CPT-1 (lipólise), cuja alta atividade está relacionada ao perfil M2. Estes efeitos foram acompanhados do aumento de citocinas pró-inflamatórias. No ambiente inflamatório (LPS) a concentração de 10 nM da toxina promoveu a marcação positiva concomitante de marcadores tanto de M1 quanto de M2. Já em 100 nM a CTX promoveu liberação paralela de citocinas pró- e anti-inflamatórias. Reunidos, os resultados nos permitem afirmar que a CTX modula funções e as vias bioenergéticas de macrófagos humanos, dependendo da concentração utilizada e do estímulo de microambiente envolvido.

The effects of sulfated hyaluronan in breast, lung and colorectal carcinoma and monocytes/macrophages cells: Its role in angiogenesis and tumor progression.

Hyaluronan (HA) is a component of the extracellular matrix (ECM) it is the main non-sulfated glycosaminoglycan able to modulate cell behavior in the healthy and tumor context. Sulfated hyaluronan (sHA) is a biomaterial derived from chemical modifications of HA, since this molecule is not naturally sulfated. The HA sulfation modifies several properties of the native molecule, acquiring antitumor properties in different cancers. In this study, we evaluated the action of sHA of ~30-60 kDa with different degrees of sulfation (0.7 sHA1 and 2.5 sHA3) on tumor cells of a breast, lung, and colorectal cancer model and its action on other cells of the tumor microenvironment, such as endothelial and monocytes/macrophage cells. Our data showed that in breast and lung tumor cells, sHA3 is able to modulate cell viability, cytotoxicity, and proliferation, but no effects were observed on colorectal cancer cells. In 3D cultures of breast and lung cancer cells, sHA3 diminished the size of the tumorsphere and modulated total HA levels. In these tumor models, treatment of monocytes/macrophages with sHA3 showed a downregulation of the expression of angiogenic factors. We also observed a decrease in endothelial cell migration and modulation of the hyaluronan-binding protein TSG-6. In the breast in vivo xenograft model, monocytes/macrophages preincubated with sHA1 or sHA3 decreased tumor vasculature, TSG-6 and HA levels. Besides, in silico analysis showed an association of TSG-6, HAS2, and IL-8 with biological processes implicated in the progression of the tumor. Taken together, our data indicate that sHA in a breast and lung tumor context is able to induce an antiangiogenic action on tumor cells as well as in monocytes/macrophages (Mo/MØ) by modulation of endothelial migration, angiogenic factors, and vessel formation.

Surface chemistry modification of silica nanoparticles alters the activation of monocytes.

Aim: Nanoparticles (NPs) interaction with immune system is a growing topic of study. Materials & methods: Bare and amine grafted silica NPs effects on monocytes/macrophages cells were analyzed by flow cytometry, MTT test and LIVE/DEAD® viability/cytotoxicity assay. Results: Bare silica NPs inhibited proliferation and induced monocyte/macrophages activation (increasing CD40/CD80 expression besides pro-inflammatory cytokines and nitrite secretion). Furthermore, silica NPs increased cell membrane damage and reduced the number of living cells. In contrast, amine grafted silica NPs did not alter these parameters. Conclusion: Cell activation properties of bare silica NPs could be hindered after grafting with amine moieties. This strategy is useful to tune the immune system stimulation by NPs or to design NPs suitable to transport therapeutic molecules.

Trojan horse monocyte-mediated delivery of conjugated polymer nanoparticles for improved photodynamic therapy of glioblastoma.

Aim: To assess monocyte-based delivery of conjugated polymer nanoparticles (CPNs) for improved photodynamic therapy (PDT) in glioblastoma (GBM). Materials & methods: Human monocyte cells (THP-1) and murine monocytes isolated from bone marrow (mBMDMs) were employed as stealth CPN carriers to penetrate into GBM spheroids and an orthotopic model of the tumor. The success of PDT, using this cell-mediated targeting strategy, was determined by its effect on the spheroids. Results: CPNs did not affect monocyte viability in the absence of light and did not show nonspecific release after cell loading. Activated monocytes incorporated CPNs in a higher proportion than monocytes in their naive state, without a loss of cellular functionality. In vitro PDT efficacy using cell-mediated delivery was superior to that using non vehiculized CPNs. Conclusion: CPN-loaded monocytes could efficiently deliver CPNs into GBM spheroids and the orthotopic model. Improved PDT in spheroids was confirmed using this delivery strategy.

A Lipidomic perspective of the action of group IIA secreted phospholipase A2 on human monocytes: lipid droplet biogenesis and activation of cytosolic phospholipase A2a

Phospholipase A2s constitute a wide group of lipid-modifying enzymes which display a variety of functions in innate immune responses. In this work, we utilized mass spectrometry-based lipidomic approaches to investigate the action of Asp-49 Ca2+-dependent secreted phospholipase A2 (sPLA2) (MT-III) and Lys-49 sPLA2 (MT-II), two group IIA phospholipase A2s isolated from the venom of the snake Bothrops asper, on human peripheral blood monocytes. MT-III is catalytically active, whereas MT-II lacks enzyme activity. A large decrease in the fatty acid content of membrane phospholipids was detected in MT III-treated monocytes. The significant diminution of the cellular content of phospholipid-bound arachidonic acid seemed to be mediated, in part, by the activation of the endogenous group IVA cytosolic phospholipase A2a. MT-III triggered the formation of triacylglycerol and cholesterol enriched in palmitic, stearic, and oleic acids, but not arachidonic acid, along with an increase in lipid droplet synthesis. Additionally, it was shown that the increased availability of arachidonic acid arising from phospholipid hydrolysis promoted abundant eicosanoid synthesis. The inactive form, MT-II, failed to produce any of the effects described above. These studies provide a complete lipidomic characterization of the monocyte response to snake venom group IIA phospholipase A2, and reveal significant connections among lipid droplet biogenesis, cell signaling and biochemical pathways that contribute to initiating the inflammatory response.

Bacterial RNA Contributes to the Down-Modulation of MHC-II Expression on Monocytes/Macrophages Diminishing CD4+ T Cell Responses.

Brucella abortus, the causative agent of brucellosis, displays many resources to evade T cell responses conducive to persist inside the host. Our laboratory has previously showed that infection of human monocytes with B. abortus down-modulates the IFN-γ-induced MHC-II expression. Brucella outer membrane lipoproteins are structural components involved in this phenomenon. Moreover, IL-6 is the soluble factor that mediated MHC-II down-regulation. Yet, the MHC-II down-regulation exerted by lipoproteins was less marked than the one observed as consequence of infection. This led us to postulate that there should be other components associated with viable bacteria that may act together with lipoproteins in order to diminish MHC-II. Our group has recently demonstrated that B. abortus RNA (PAMP related to pathogens' viability or vita-PAMP) is involved in MHC-I down-regulation. Therefore, in this study we investigated if B. abortus RNA could be contributing to the down-regulation of MHC-II. This PAMP significantly down-modulated the IFN-γ-induced MHC-II surface expression on THP-1 cells as well as in primary human monocytes and murine bone marrow macrophages. The expression of other molecules up-regulated by IFN-γ (such as co-stimulatory molecules) was stimulated on monocytes treated with B. abortus RNA. This result shows that this PAMP does not alter all IFN-γ-induced molecules globally. We also showed that other bacterial and parasitic RNAs caused MHC-II surface expression down-modulation indicating that this phenomenon is not restricted to B. abortus. Moreover, completely degraded RNA was also able to reproduce the phenomenon. MHC-II down-regulation on monocytes treated with RNA and L-Omp19 (a prototypical lipoprotein of B. abortus) was more pronounced than in monocytes stimulated with both components separately. We also demonstrated that B. abortus RNA along with its lipoproteins decrease MHC-II surface expression predominantly by a mechanism of inhibition of MHC-II expression. Regarding the signaling pathway, we demonstrated that IL-6 is a soluble factor implicated in B. abortus RNA and lipoproteins-triggered MHC-II surface down-regulation. Finally, CD4+ T cells functionality was affected as macrophages treated with these components showed lower antigen presentation capacity. Therefore, B. abortus RNA and lipoproteins are two PAMPs that contribute to MHC-II down-regulation on monocytes/macrophages diminishing CD4+ T cell responses.

Hyaluronan preconditioning of monocytes/macrophages affects their angiogenic behavior and regulation of TSG-6 expression in a tumor type-specific manner.

Hyaluronan is a glycosaminoglycan normally present in the extracellular matrix in most tissues. Hyaluronan is a crucial player in many processes associated with cancer, such as angiogenesis, invasion, and metastasis. However, little has been reported regarding the action of hyaluronan on monocytes/macrophages (Mo/MØ) in tumor angiogenesis and its consequences on tumor development. In the present study, we investigated the effects of hyaluronan of different sizes on human Mo/MØ angiogenic behavior in colorectal and breast carcinoma. In vitro, the treatment of Mo/MØ with lysates and conditioned media from a breast but not from colorectal carcinoma cell line plus high-molecular weight hyaluronan induced: (a) an increased expression of angiogenic factors VEGF, IL-8, FGF-2, and MMP-2, (b) an increased endothelial cell migration, and (c) a differential expression of hyaluronan-binding protein TSG-6. Similar results were observed in Mo/MØ derived from breast cancer patients treated with tumor lysates. Besides, macrophages primed with high-molecular weight hyaluronan and inoculated in human breast cancer xenograft tumor increased blood vessel formation and diminished TSG-6 levels. In contrast, the effects triggered by high-molecular weight hyaluronan on Mo/MØ in breast cancer context were not observed in the context of colorectal carcinoma. Taken together, these results indicate that the effect of high-molecular weight hyaluronan as an inductor of the angiogenic behavior of macrophages in breast tumor context is in part consequence of the presence of TSG-6.

Platelets Promote Brucella abortus Monocyte Invasion by Establishing Complexes With Monocytes.

Brucellosis is an infectious disease elicited by bacteria of the genus Brucella. Platelets have been extensively described as mediators of hemostasis and responsible for maintaining vascular integrity. Nevertheless, they have been recently involved in the modulation of innate and adaptive immune responses. Although many interactions have been described between Brucella abortus and monocytes/macrophages, the role of platelets during monocyte/macrophage infection by these bacteria remained unknown. The aim of this study was to investigate the role of platelets in the immune response against B. abortus. We first focused on the possible interactions between B. abortus and platelets. Bacteria were able to directly interact with platelets. Moreover, this interaction triggered platelet activation, measured as fibrinogen binding and P-selectin expression. We further investigated whether platelets were involved in Brucella-mediated monocyte/macrophage early infection. The presence of platelets promoted the invasion of monocytes/macrophages by B. abortus. Moreover, platelets established complexes with infected monocytes/macrophages as a result of a carrier function elicited by platelets. We also evaluated the ability of platelets to modulate functional aspects of monocytes in the context of the infection. The presence of platelets during monocyte infection enhanced IL-1ß, TNF-α, IL-8, and MCP-1 secretion while it inhibited the secretion of IL-10. At the same time, platelets increased the expression of CD54 (ICAM-1) and CD40. Furthermore, we showed that soluble factors released by B. abortus-activated platelets, such as soluble CD40L, platelet factor 4, platelet-activating factor, and thromboxane A2, were involved in CD54 induction. Overall, our results indicate that platelets can directly sense and react to B. abortus presence and modulate B. abortus-mediated infection of monocytes/macrophages increasing their pro-inflammatory capacity, which could promote the resolution of the infection.

Evidence of size-dependent effect of silica micro- and nano-particles on basal and specialized monocyte functions.

AIM: To analyze the effect of silica particles on monocyte/macrophage functions. MATERIALS & METHODS: Silica micro- and nanoparticles were obtained by the Stöber method. Their effect on monocyte/macrophage proliferation, activation, membrane integrity and metabolic activity were determined. RESULTS: Silica particles inhibit cell proliferation while 10 nm nanoparticles (NPs) did not affect it. Similarly, silica particles induced strong cell activation. However, 10 nm NPs do not alter IL-12 or nitrite levels. Furthermore, bigger NPs and microparticles increase cell membrane damage and reduce the number of living cells but smallest NPs (10 and 240 nm) did not. CONCLUSION: Cell activation properties of silica particles could be useful tools for immune stimulation therapy, while 10 nm NPs would be suitable for molecule transportation.

Brucella abortus down-regulates MHC class II by the IL-6-dependent inhibition of CIITA through the downmodulation of IFN regulatory factor-1 (IRF-1).

Brucella abortus is an intracellular pathogen capable of surviving inside of macrophages. The success of B. abortus as a chronic pathogen relies on its ability to orchestrate different strategies to evade the adaptive CD4+ T cell responses that it elicits. Previously, we demonstrated that B. abortus inhibits the IFN-γ-induced surface expression of MHC class II (MHC-II) molecules on human monocytes, and this phenomenon correlated with a reduction in antigen presentation. However, the molecular mechanisms, whereby B. abortus is able to down-regulate the expression of MHC-II, remained to be elucidated. In this study, we demonstrated that B. abortus infection inhibits the IFN-γ-induced transcription of MHC-II, transactivator (CIITA) and MHC-II genes. Accordingly, we observed that the synthesis of MHC-II proteins was also diminished. B. abortus was not only able to reduce the expression of mature MHC-II, but it also inhibited the expression of invariant chain (Ii)-associated immature MHC-II molecules. Outer membrane protein 19 (Omp19), a prototypical B. abortus lipoprotein, diminished the expression of MHC-II and CIITA transcripts to the same extent as B. abortus infection. IL-6 contributes to these down-regulatory phenomena. In addition, B. abortus and its lipoproteins, through IL-6 secretion, induced the transcription of the negative regulators of IFN-γ signaling, suppressor of cytokine signaling (SOCS)-1 and -3, without interfering with STAT1 activation. Yet, B. abortus lipoproteins via IL-6 inhibit the expression of IFN regulatory factor 1 (IRF-1), a critical regulatory transcription factor for CIITA induction. Overall, these results indicate that B. abortus inhibits the expression of MHC-II molecules at very early points in their synthesis and in this way, may prevent recognition by T cells establishing a chronic infection.