(D-142) Macrophage Polarization: Investigating NF-κB Pathway Signaling in Response to Cytokine Stimulation

Macrophages are a class of immune cells involved in almost every disease and present in all human tissues. These immune responders represent attractive targets for many modern therapies due to their plasticity and diverse range of functions [1]. Two broad groupings of macrophage phenotype have been described: M1-like which arise in pro-inflammatory conditions, and M2-like which arise in pro-healing conditions [2]. Transition into these two phases from the undifferentiated M0 stage is termed polarization [2].  Inflammatory responses play key roles in the development and persistence of many disease states [3]. A key signaling pathway in macrophages responsible for the onset of inflammation as well as a number of other functions is the NFκB pathway [4]. The initiation of this pathway is affected by a number of small, secreted proteins released by cells have specific effects on intercellular interactions and communications called cytokines. In macrophages, IFN-γ, TNF-α, and IL-12 are the primary cytokines involved in polarization. [5]. Due to macrophage plasticity and functional diversity, it is important to characterize the mechanisms behind their polarization.

In this study, I used a RAW264.7 model mouse macrophage cell line modified with an mCherry fluorescent protein reporter inserted at the NFκB promoter region. After stimulating RAW264.7s with IFN-γ, TNF-α, and IL-12, fluorescence imaging was conducted on a confocal imaging system every 24 hours for 3 days to assess the effects of cytokine stimulation on NFκB expression.

RAW 264.7 model mouse macrophages were cultured to approximately 70% confluency in Dulbecco’s Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum and 1% PenStrep. For each of four experimental groups (IFN-γ, TNF-α, IL-12, and negative control), four MatTek dishes were designated for set timepoints (0, 24, 48, 72hr). In each dish, 250,000 RAW 264.7 cells in 1mL DMEM were plated. After a 24 hour incubation period, RAW 264.7 macrophages were stimulated with 10ng/mL recombinant mouse IFN-γ protein, 50ng/mL recombinant mouse TNF-α protein, and 10 ng/mL recombinant mouse IL-12 protein, all obtained from RndSystems, in 2mL DMEM. 1mL DMEM with no cytokine addition was introduced to the negative control group. At this point (0 hours), cells in each plate were excited with a laser at 587nm, emission was recorded at 610 nm (excitation and emission peaks for mCherry), and images were taken simultaneously in multiple fields of view, on a FluoView FV10i-LIV system. At 24, 48, and 72 hours, images were taken with the same parameters. After imaging, cells were fixed and permeabilized with 10% formalin and 10% Triton-X. After incubation in Brilliant Violet 421 anti-mouse CD80 antibody (excitation 405nm, emission 421nm) purchased from BioLegend, diluted in 0.17% BSA, M1 polarization was confirmed on the FluoView FV10i-LIV system.

 A small basal level of fluorescence was observed in cells with no cytokine addition. Considerably greater fluorescence was observed in other groups treated with IFN-γ, TNF-α, and IL-12. The largest increase in fluorescence of all cytokine treated groups was observed in the first 24 hours after treatment. Some increases were observed in subsequent measurements, however, these were also observed in the negative control group and may be attributed to responses caused by growth and crowding. Immunostaining for CD80 ligand, a key contributor in macrophage activation, confirmed M1 polarization of test groups.

Image analysis will be conducted with Fiji (ImageJ) to characterize the fluorescence intensity of 100 cells from at least 3 different fields of view for each timepoint of each experimental group. In the presentation of the outcomes of this REU project, this metric will be used to quantify the results of acquired images.

In this study, the role of NFκB pathway signaling macrophage polarization was investigated. After treating RAW 264.7 murine macrophages with IFN-γ, TNF-α, and IL-12, a notable increase in NFκB expression was observed, indicating a link to the activation of the pro-inflammatory M1-like phenotype. The peak fluorescence was observed at 24 hours post-stimulation, suggesting a rapid response to the cytokines. These results shed light on the mechanisms behind macrophage polarization, offering potential insights for targeted therapeutic approaches.

(1)   https://www.nature.com/articles/nature12034#Sec12
(2)   https://www.annualreviews.org/doi/abs/10.1146/annurev-physiol-022516-034339 Polarization
(3) https://europepmc.org/article/med/12617561
(4) https://europepmc.org/article/med/9238509
(5) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785020/