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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session C

Poster I7 - Novel Method to Assess Macrophage Phenotypes Using Eluted Cell Media

Friday, October 25, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

FM

Furqan Mahdi

Undergraute Biomedical Engineering Student
University of Nebraska Lincoln
Lincoln, Nebraska, United States

Co-Author(s)

DL

David Lillyman

Graduate Student
University of Nebraska Lincoln, United States
KN

Kayla Ney

Graduate Student
University of Nebraska Lincoln, United States

Primary Investigator(s)

RW

Rebecca Wachs, PhD (she/her/hers)

Associated Professor
Arizona State University, United States

Introduction: Macrophages are a crucial part of the immune system [1]. They defend against pathogens, cause inflammation, clear debris, and promote tissue repair [1]. Due to their wide range of functions, macrophages exhibit plasticity tailored to specific responses depending on the microenvironment of the macrophages [2]. The plasticity trait also allows macrophages to have a wide range of phenotypes [3]. Despite the spectrum of macrophage phenotypes, two distinct phenotypes are often focused on, M1 and M2 [3]. The M1 macrophages are known to be pro-inflammatory, they respond to injury or infection [4]. The M2 macrophages are anti-inflammatory macrophages that function as tissue repair macrophages [5]. Due to their plasticity and wide range of phenotypes spectrum, categorizing macrophage phenotypes is challenging to perform in vitro. Currently, immunocytochemistry (ICC) is the gold standard for assessing the polarization of macrophages. When a macrophage adopts the M1 phenotype, it produces nitric oxide synthase (iNOS), which, in turn, catalyzes the digestion of the amino acid arginine into nitrite [6].
Additionally, M2 macrophages produce arginase (Arg1), an enzyme that converts arginine into urea [6]. The ICC method stains for iNOS and Arg1. However, ICC is a time-intensive process, spanning two days, and a costly method. Furthermore, ICC is an endpoint assay requiring cell fixation, limiting further investigation of life cells. Due to the differential processing of arginine between M1 and M2, we hypothesized that we could effectively assess macrophage polarization using commercialized kits to measure nitrite and urea production in eluted cell media.

Materials and

Methods: Primary bone marrow cells were harvested from adult male and female Sprague Dawley rats (n=4). The harvested bone marrow cells were cultured and differentiated into macrophages (M0) for 6 days using macrophage colony-stimulating factor (MCSF). After differentiation, the macrophages were polarized into M1-like and M2-like phenotypes using interferon-γ (IFN-γ) and lipopolysaccharide (LPS) or interleukin-4 (IL-4) and interleukin-10 (IL-10), respectively for 24 hours. After the 24-hour treatment, the eluted cell media was collected to measure the production of nitrite and urea using commercialized kits, and the cells were fixed to perform ICC. Fixed cells were stained with primary antibodies for iNOS (M1-like) or Arg1 (M2-like) and secondary fluorescent antibodies. Cell staining was imaged using a Cytation1 reader and analyzed using ImageJ. The nitrite and urea concentration levels in eluted media were measured using a microplate reader. Correlations between iNOS and nitrite, Arg1, and urea were performed to validate the nitrite assay to assess macrophage phenotype. Ratios of relative staining (ICC) or concentrations (metabolite assays) were performed to determine “M1 likeness” and “M2 likeness”. Nitrite and urea production as well as ICC staining intensity were assessed for normality which showed a non-normal distribution, therefore a Kruskal-Wallis test was performed on the samples followed by a Dunn’s post hoc test. All data are presented as mean ± standard deviation and results were considered statistically significant when p < 0.05.

Results, Conclusions, and Discussions:

Results:
The ICC staining for iNOS showed a significant iNOS expression in M1-like macrophages when compared to M0 (Fig. 1A). The novel method of using nitrite also showed a significant increase in nitrite concentration in the M1-like macrophages when compared to M0 (Fig. 1B). A linear correlation showed a significant correlation between iNOS staining intensity and nitrite concentration levels (Fig. 1C). The ICC staining for Arg1 revealed a significant expression in M2-like macrophages when compared to M0 (Fig. 2A). Similarly, the novel method of using urea kits to measure urea production in eluted media showed significantly higher levels of urea production in M2-like macrophages compared to M0 (Fig. 2B). The linear correlation analysis between Arg1 staining and urea levels also showed significant results (Fig. 2C). The relative ratios of iNOS to Arg1 and nitrite to urea showed that the cells are M1-like macrophages (Fig. 3A-B). The relative ratios of Arg1 to iNOS and urea to nitrite showed that the cells are more M2-like macrophages (Fig. 3C-D).

Conclusion and

Discussion:
Differentiation between macrophage phenotypes such as M1 and M2 is necessary for understanding immune responses in disease models and biomaterial integration. In this study, we validated a rapid and cost-effective method to assess macrophage polarization using nitrite and urea measurements in macrophage-secreted culture media, offering a non-destructive alternative to traditional ICC methods. One of the significant advantages of the proposed method is its applicability in biomaterial research. Cells that are embedded or infiltrated into the biomaterials are hard to visualize. To be able to properly visualize the cells immunohistochemistry (IHC) staining is needed in the biomaterial which requires a destruction of the biomaterial that can limit any further investigation.

Acknowledgements (Optional):