(D-159) Characterization of macrophage polarization states following shear stress exposure

Introduction:: Macrophages can in some cases make up 50% of the tumor mass and tend to correlate with poor patient prognosis [1]. Since macrophages make up a considerable portion of the tumor mass, utilizing these populations of cells to create an immunosupportive environment can help sensitize tumors to other immunotherapies by increasing lymphocyte infiltration. Macrophage polarization exists on a spectrum where the endpoints make up two distinct populations of macrophages: M1 and M2 macrophages [2]. During the normal course of infection, macrophages are often one of the first lines of defense against a pathogen, and exhibit cell surface markers that facilitate antigen presentation and production of anti-inflammatory cytokines such as IFN-γ and IL-6 [3]. Once the immune response attenuates and the pathogen is cleared, macrophages begin to shift their phenotype to exhibit cell surface markers that assist in the phagocytic function of macrophages and secrete anti-inflammatory cytokines such as IL-10 and TGF-β [3]. The current thought is that the majority of macrophages that reside in a tumor has a M2 macrophage phenotype and help create an immunosuppressive environment [1]. Therefore, shifting the M2 macrophages at the tumor site toward an M1 macrophage phenotype may help create a more immunosupportive microenvironment at the site of the tumor leading to better prognosis. We propose that exposure of macrophages to shear stress re-educates the macrophages to a M1-like pro-inflammatory phenotype and deserves further study.


Materials and Methods:: Thp-1 cells, a monocyte cell line isolated from a 3-year-old leukemic patient, were expanded in culture. The monocytes were plated in a non-treated 6 well plate at a concentration of 33,300 cells/mL and incubated overnight. After 24 hours of rest, monocytes were exposed to 150 nM of phorbol 12-myristate 13-acetate (PMA) for 24 hours to differentiate the monocytes into unpolarized macrophages (M0). Following 24 hours of PMA exposure, it was confirmed that the cells have adhered to the bottom of the well plate and the PMA-media removed and replaced with fresh media. After 24 hours of rest, macrophages were then polarized to either a M1 or M2a phenotype. To generate the M1 phenotype, Thp-1 macrophages were exposed to 40 ng/mL of IFN-γ and 150 ng/mL of LPS for 24 hours [3]. The M2 phenotype was generated by exposing the Thp-1 macrophages to 20 ng/mL of IL-4 and 20 ng/mL of IL-13 for 48 hours [3]. Exposing unpolarized macrophages to anti-inflammatory cytokines, such as IL-4, generates the M2a subpopulation, which are the alternatively activated wound healing macrophages. CD86 was used as an indicator for the M1 population, CD206 as a marker for the M2a population, and CD68 as a general macrophage marker for the M0 population.


Results, Conclusions, and Discussions:: Results

It was found that following shear stress exposure, the macrophages become smaller and more granular (Fig. 1). Following 24 hours post fluid shear stress, monocytes were found to express more actin and began to form clusters (Fig. 1). It was confirmed that M1 macrophages preferentially express CD86 and secrete CXCR10, which is indicative of pro-inflammatory macrophages. It was also confirmed that M2 macrophages preferentially express CD206, and M0 macrophages do not express CD206 or CD86 but rather preferentially express CD68 (Fig. 1). Following shear stress exposure, little difference was observed between CD86 and CD206 expression (Fig. 1).

Discussion and Conclusions

It was concluded that M1 and M2 macrophages have distinct surface marker profiles that potentially tie in with the effector functions of macrophages. Once exposed to shear stress, the macrophages become smaller and more granular, which is indicative of a change in the properties within the cell. Following 24 hours after exposure to shear stress, we saw a slight increase in CD86 expression within our M2 macrophages, which could indicate an emergence of this M2b subpopulation. Shear stress could have beneficial effects in the re-education of macrophages, exhibited by the slight increase in CD86 expression within the M2 population. Following shear stress, monocytes were seen to become adherent, which is used as an indicator of the emergence of the M0 macrophage population. In addition to the sheared monocytes becoming adherent, they also become more granular and smaller, as is true for M2 macrophages, and also seem to express more actin. These morphological changes brought on by exposure to shear stress could be indicative of a shift towards a M0-state without the use of PMA to stimulate the differentiation of monocytes to a macrophage-like state.


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References (Optional): : References

1. Poh, A.R. and Ernst, M. (2018) “Targeting macrophages in cancer: From bench to bedside,” Frontiers in Oncology, 8. https://doi.org/10.3389/fonc.2018.00049.

2. Pérez, S. and Rius-Pérez, S. (2022) “Macrophage polarization and reprogramming in acute inflammation: A redox perspective,” Antioxidants, 11(7), p. 1394. https://doi.org/10.3390/antiox11071394.

3. Yao, Y., Xu, X.-H. and Jin, L. (2019) “Macrophage polarization in physiological and pathological pregnancy,” Frontiers in Immunology, 10. https://doi.org/10.3389/fimmu.2019.00792.