Protocol for localized macrophage stimulation with small-molecule TLR agonist via fluidic force microscopy.

Here, we present a protocol to deliver nanoliter volumes of Toll-like receptor (TLR) agonist onto a culture of nuclear factor κB (NF-κB) reporter macrophages using fluidic force microscopy and a micron-scale probe. We describe steps for quantifying the dose of agonist by modeling their diffusion with experimental inputs. We then detail procedures for quantifying and categorizing macrophage responses to individual and varied doses and combining agonist concentration and macrophage response to analyze the NF-κB response to localized TLR stimulation. For complete details on the use and execution of this protocol, please refer to Mulder et al. (2024).1.

Moderate mechanical stress suppresses chondrocyte ferroptosis in osteoarthritis by regulating NF-κB p65/GPX4 signaling pathway.

Ferroptosis is a recently identified form of programmed cell death that plays an important role in the pathophysiological process of osteoarthritis (OA). Herein, we investigated the protective effect of moderate mechanical stress on chondrocyte ferroptosis and further revealed the internal molecular mechanism. Intra-articular injection of sodium iodoacetate (MIA) was conducted to induce the rat model of OA in vivo, meanwhile, interleukin-1 beta (IL-1ß) was treated to chondrocytes to induce the OA cell model in vitro. The OA phenotype was analyzed by histology and microcomputed tomography, the ferroptosis was analyzed by transmission electron microscope and immunofluorescence. The expression of ferroptosis and cartilage metabolism-related factors was analyzed by immunohistochemical and Western blot. Animal experiments revealed that moderate-intensity treadmill exercise could effectively reduce chondrocyte ferroptosis and cartilage matrix degradation in MIA-induced OA rats. Cell experiments showed that 4-h cyclic tensile strain intervention could activate Nrf2 and inhibit the NF-κB signaling pathway, increase the expression of Col2a1, GPX4, and SLC7A11, decrease the expression of MMP13 and P53, thereby restraining IL-1ß-induced chondrocyte ferroptosis and degeneration. Inhibition of NF-κB signaling pathway relieved the chondrocyte ferroptosis and degeneration. Meanwhile, overexpression of NF-κB by recombinant lentivirus reversed the positive effect of CTS on chondrocytes. Moderate mechanical stress could activate the Nrf2 antioxidant system, inhibit the NF-κB p65 signaling pathway, and inhibit chondrocyte ferroptosis and cartilage matrix degradation by regulating P53, SLC7A11, and GPX4.

NFkB y su importancia en artritis e inflamación / NFkB and its importance in arthritis and inflammation

El factor de transcripción NFkB tiene una participación muy importante en el desarrollo y mantención de una serie de patologías humanas, principalmente aquellas con un componente inflamatorio, como la artritis reumatoide (AR). Al mismo tiempo participa en procesos tan diversos como la regulación de la respuesta inmune y el desarrollo embrionario. Una mejor comprensión de los mecanismos y funciones de NFkB permitiría el desarrollo de drogas específicas y efectivas para el tratamiento de patologías inflamatorias y autoinmunes, tratando de no interferir con las funciones normales de este sistema.

Transcription factor NFkB has an important role in development and maintenance of a lot of human pathologies, mainly those with an inflammatory component, for example rheumatoid arthritis (RA). At the same time, it participates in processes as diverse as development and immune response. A better understanding of NFkB mechanisms and functions will allow the development of more specific and effective drugs for the treatment of inflammatory and autoimmune disorders, without interfering with normal functions of this system.

Role of nuclear factor kappa B and reactive oxygen species in the tumor necrosis factor-a-induced epithelial-mesenchymal transition of MCF-7 cells

The microenvironment of the tumor plays an important role in facilitating cancer progression and activating dormant cancer cells. Most tumors are infiltrated with inflammatory cells which secrete cytokines such as tumor necrosis factor-alpha (TNF-alpha). To evaluate the role of TNF-alpha in the development of cancer we studied its effects on cell migration with a migration assay. The migrating cell number in TNF-alpha-treated group is about 2-fold of that of the control group. Accordingly, the expression of E-cadherin was decreased and the expression of vimentin was increased upon TNF-alpha treatment. These results showed that TNF-alpha can promote epithelial-mesenchymal transition (EMT) of MCF-7 cells. Further, we found that the expression of Snail, an important transcription factor in EMT, was increased in this process, which is inhibited by the nuclear factor kappa B (NFkB) inhibitor aspirin while not affected by the reactive oxygen species (ROS) scavenger N-acetyl cysteine. Consistently, specific inhibition of NFkB by the mutant IkBalpha also blocked the TNF-alpha-induced upregulation of Snail promoter activity. Thus, the activation of NFkB, which causes an increase in the expression of the transcription factor Snail is essential in the TNF-alpha-induced EMT. ROS caused by TNF-alpha seemed to play a minor role in the TNF-alpha-induced EMT of MCF-7 cells, though ROS per se can promote EMT. These findings suggest that different mechanisms might be responsible for TNF-alpha - and ROS-induced EMT, indicating the need for different strategies for the prevention of tumor metastasis induced by different stimuli.

Nuclear factor-kB in thyroid carcinogenesis and progression: a novel therapeutic target for advanced thyroid cancer

Apoptosis is an essential physiological process of elimination of destined cells during the development and differentiation or after damage from external stresses such as ionizing radiation or chemotherapeutic agents. Disruption of apoptosis is proved to cause various diseases including cancer. Among numerous molecules involved in diverse anti- or pro-apoptotic signaling pathways, NF-kappaB is one of the key factors controlling anti-apoptotic responses. Its anti-apoptotic effect is thought to be mediated through not only transcriptional activation of dependent genes but also by crosstalking with the JNK pathway. Oncogenic proteins such as Ret/PTC, Ras and BRAF can induce NF-kappaB activation making it an important change in thyroid cancer. A number of specific or non-specific NF-kappaB inhibitors have been tried to take over the cascade in in vitro and in vivo experiments. These agents can induce massive apoptosis especially in combination with radio- or chemotherapy. Current results suggest that the inhibition of the NF-kappaB may be a promising strategy for advanced thyroid cancer treatment but further investigations are warranted to develop specific and clinically effective NF-kappaB inhibitors in future.

A apoptose é um processo fisiológico essencial destinado a eliminar células durante o desenvolvimento e diferenciação ou após danos decorrentes de estresses externos com a radiação ionizante ou agentes quimioterápicos. Distúrbios na apoptose têm sido demonstrados como causadores de várias doenças, incluindo câncer. Entre as inúmeras moléculas envolvidas nas várias vias de sinalização anti- ou pró-apoptoticas, NF-kapaB é um dos fatores-chave que controlam as respostas anti-apoptóticas. Acredita-se que seu efeito anti-apoptótico seja mediado não apenas pela ativação transcricional de genes dependentes mas também por crosstalking com a via JNK. Proteínas oncogênicas como Ret/PTC, Ras e BRAF podem induzir ativação de NF-kapaB promovendo importante transformação no câncer da tireóide. Uma série de inibidores específicos e não-específicos do NF-kapaB tem sido usada em experimentos in vitro e in vivo procurando inibir a cascata. Esses agentes podem induzir apoptose maciça especialmente em combinação com radio ou quimioterapia. Resultados atuais sugerem que a inibição de NF-kapaB pode ser uma estratégia promissora no tratamento do câncer da tireóide avançado, mas novas investigações são necessárias para desenvolver inibidores específicos e clinicamente efetivos do NF-kapaB.

Papel inmunológico de la progesterona en el mantenimiento del embarazo / Immunological role of progesterone in the maintenance of pregnancy

Progesterone is an essential hormone for pregnancy maintenance. This hormone acts by binding to its intracellular receptor or by rapid non-genomic actions to regulate a wide variety of biological functions in the feto-placental unit. Progesterone regulates blastocyst implantation and placental development by inducing immunosupression through type Th2 cytokines secretion. This review summarizes current research about the role of progesterone as critical regulator of expression and secretion of cytokines by T-cell and other placental cells.

La progesterona es una hormona esteroide muy versátil y esencial para el mantenimiento del embarazo. El principal mecanismo de acción de la progesterona es el clásico, vía receptor intracelular, regulando diversas funciones, aspectos celulares y vías moleculares implicadas en el proceso de la implantación. Asimismo existen mecanismos adicionales que no dependen de la interacción del complejo hormona receptor con la maquinaria transcripcional y que son capaces de regular rápidamente cascadas de señalización que determinarán la respuesta de la célula. En particular se ha demostrado que la progesterona ejerce efectos inmunosupresores durante la gestación al favorecer la secreción de citocinas de tipo Th2 por los linfocitos T, evento importante para regular el sistema inmunológico materno y evitar el rechazo de la placenta. El objetivo de esta revisión se centra en analizar la influencia de la progesterona en la interfase materno-fetal sobre la expresión y secreción de citocinas por las células T y no T como es el caso del trofoblasto.

El papel del factor de transcripción NF-κB en la célula cardíaca / Role of the transcription factor NF-κB in the cardiac cell

Para la biología de hoy las vías de señalización intracelular que controlan los procesos entre la vida y la muerte celular son de gran interés. Al respecto, el NF-κB destaca como un factor de transcripción decisivo de respuesta rápida que participa en la activación de las vías de señalización de la muerte celular programada. Lo relevante es que sus efectos tienen consecuencias en el desarrollo normal y/o la homeostasis en muchas células o tejidos, que incluyen entre otros al sistema inmune, los folículos capilares, apéndices epidermales, el riñon y el sistema nervioso. En esta revisión analizamos el papel central que juega el factor de transcripción NF-κB en el funcionamiento normal de la célula cardíaca y sus implicaciones en algunas de las patologías cardíacas más frecuentes como: el daño por isquemia-reperfusión, la isquemia precondicionada, la hipertrofia, la aterosclerosis, y el paro cardíaco. El NF-κB comúnmente funciona como un agente citoprotector, aunque hay algunos casos en los cuales resulta ser pro-apoptótico dependiendo del estímulo y del contexto celular. Se han logrado avances significativos a nivel molecular, que han permitido entender su modo de acción y el papel interactivo que juega con otros factores claves. Estos estudios han identificado muchos genes anti-apoptóticos y pro-apoptóticos regulados por la actividad del NF-κB abriendo novedosas aproximaciones que se pueden hacer sobre sus efectos en el desarrollo de patologías cardíacas.

The signaling pathways that control the life-death switch of a cell are a prime interest in Modern Biology. To this respect, NF-κB has emerged as a decisive transcription factor in the cell's response to apoptotic challenge and its effects on apoptosis have far-reaching consequences for normal development and/or homeostasis in many cells and tissues, including the immune system, hair follicles, and epidermal appendages, the liver, and nervous system. In this review we analyze the pivotal role of the transcription factor NF-κB in the normal functioning of the cardiac cell and its implication on some of the most frequent cardiac pathologies, such as ischemia-reperfusion injury, ischemic precondition, hypertrophy, atherosclerosis and cardiac arrest. While NF-κB is commonly found to be cytoprotective, there are a number of instances where it is proapoptotic depending on the inducing stimulus and the cell context. Significant progress has been made in understanding its mode of action and its interplay with other key factors. These studies identified many anti- and pro-apoptotic NF-κB regulated genes that mediate its activity, these important new insights fuel hope that novel approaches will be developed to control the effects of NF-κB in cardiac pathologies.

The maternal JAK/STAT pathway of Drosophila regulates embryonic dorsal-ventral patterning

Activation of NFkappaB plays a pivotal role in many cellular processes such as inflammation, proliferation and apoptosis. In Drosophila, nuclear translocation of the NFkappaB-related transcription factor Dorsal is spatially regulated in order to subdivide the embryo into three primary dorsal-ventral (DV) domains: the ventral presumptive mesoderm, the lateral neuroectoderm and the dorsal ectoderm. Ventral activation of the Toll receptor induces degradation of the IkappaB-related inhibitor Cactus, liberating Dorsal for nuclear translocation. In addition, other pathways have been suggested to regulate Dorsal. Signaling through the maternal BMP member Decapentaplegic (Dpp) inhibits Dorsal translocation along a pathway parallel to and independent of Toll. In the present study, we show for the first time that the maternal JAK/STAT pathway also regulates embryonic DV patterning. Null alleles of loci coding for elements of the JAK/STAT pathway, hopscotch (hop), marelle (mrl) and zimp (zimp), modify zygotic expression along the DV axis. Genetic analysis suggests that the JAK kinase Hop, most similar to vertebrate JAK2, may modify signals downstream of Dpp. In addition, an activated form of Hop results in increased levels of Cactus and Dorsal proteins, modifying the Dorsal/Cactus ratio and consequently DV patterning. These results indicate that different maternal signals mediated by the Toll, BMP and JAK/STAT pathways may converge to regulate NFkappaB activity in Drosophila.