Differential Effects of Fluid Shear Stress on Macrophage Functionality and Polarization

Introduction: Macrophages can encompass up to 50% of tumor mass, and their presence tends to correlate with poor patient prognosis [1]. Macrophages within the tumor microenvironment tend to be anti-inflammatory, which aids in creating an immunosuppressive microenvironment and promoting tumor progression. Macrophage polarization exists on a spectrum where the endpoints incorporate two distinct populations of macrophages: M1 and M2 [2]. During a perfunctory 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 pro-inflammatory cytokines such as IFN- γ, TNF-⍺, IL-1, and IL-6 [3]. Once the immune response attenuates and the pathogen is cleared, macrophages shift their phenotype toward an M2 phenotype that exhibits cell surface markers that assist in the phagocytic function of macrophages and secretion of anti-inflammatory cytokines such as IL-10 and TGF-β [3]. Exposing M0 macrophages to anti-inflammatory cytokines, such as IL-4, generates the M2 subpopulation, which are the alternatively activated wound-healing macrophages. The current thought is that the majority of macrophages that reside in a tumor have an M2 macrophage phenotype and help create an immunosuppressive environment [1]. Therefore,understanding the dynamic effects of fluid shear stress (FSS) on macrophage polarization states can provide valuable insight into how tumor-associated macrophages function as they enter the bloodstream during metastasis. We propose that exposure of macrophages to shear stress throughout the differentiation and polarization process will exhibit a diverse effect on macrophage polarization dependent on their initial state.

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 non-treated 6-well plates at 66,667 cells/mL concentration 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 had adhered to the bottom of the well plate, and the PMA-media was removed and replaced with fresh media. After 24 hours of rest, macrophages were polarized to either the M1 or M2 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/mLs of IL-4 and IL-13 for 48 hours. Monocytes, M0, M1, and M2 macrophages were exposed to fluid shear stress for 1 hr in a cone and plate viscometer and allowed to incubate for 24 hr. Following the incubation period, cells were prepped for imaging or phagocytosis assays and the media frozen for ELISA assays to measure NFkB phosphorylation, phagocycic activity, and cytokine secretion.

Results, Conclusions, and Discussions: All macrophages showed increased phosphorylation of NFkB and relatively similar levels of actin when compared to their respective sheared groups(Fig1A,B,C). Sheared monocytes and M2 macrophages showed higher phagocytic activity when compared to static conditions, however M1 and M0 macrophages showed no change in phagocytic activity following FSS exposure(Fig.1D,E). After exposure of a calcium chelator, EGTA, during FSS exposure, M2-sheared macrophages were shown to exhibit a significant decrease in phagocytic activity compared to sheared M2 macrophages without EGTA and static conditions. M1 macrophages secreted more TNF-⍺ and IFN-γ compared to M0 and M2 macrophages, sheared M1 macrophages secreted more IFN-γ and similar levels of TNF-⍺. M0 and M2-sheared macrophage groups secreted more TNF-⍺ when compared to their static counterparts. Shear stress can affect subpopulations of macrophages differently, which indicates that FSS could be affecting macrophages within circulating tumor clusters in different ways that may make one population more successful in forming metastatic tumors. In the case of M2 macrophages, the addition of a calcium chelator negated the effects of FSS and indicates that phagocytic activity could be affected through calcium signaling. Shear stress affects M1 macrophages differently, by enhancing type-1 cytokine production, like IFN-γ and TNF-⍺, and enhancing NFkB phosphorylation, while FSS does not affect M1 phagocytic activity. This indicates that it may enhance the M1 macrophage effector functions through creating a more immunosupportive microenvironment. Interestingly, shear stress affects unpolarized macrophages by increasing the secretion of pro-inflammatory cytokine TNF-⍺ but not IFN-γ and does not affect phagocytic activity and has similarly low levels of phagocytic activity that is comparable to M1 macrophages. This may indicate that shear stress helps to polarize unpolarized macrophages to a M1-like phenotype rather than an M2-like phenotype. Conversely, sheared M2 macrophages may repolarize to a different macrophage subpopulation that retains M2 effector function but also secretes pro-inflammatory cytokines, like TNF-⍺. Ongoing research is addressing the effects of macrophage polarization as it relates to cancer metastasis by co-culturing macrophages and cancer cells to understand the effects macrophages have on cancer cell survival and model the circulating tumor clusters that are found in the bloodstream of cancer patients.

Acknowledgements (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.