L5 - Cyclical Shear Stress Selectively Primes Monocytes Toward a Hybrid Immunomodulatory State

Introduction: : Monocytes circulate in peripheral blood for several days and are the precursor to essential cells that control tumor progression, such as macrophages and dendritic cells, to orchestrate immune reactions in the tumor microenvironment that can control disease outcome [1]. Monocytes can be classified into three main subtypes based on surface expression of CD14 and CD16: classical, intermediate, and non-classical monocytes. Classical monocytes, defined as CD14++/CD16+, comprise the majority of the monocyte population and main effector function to be phagocytosis and secretion of IL-10, which tend to function as anti-inflammatory macrophages [2]. Non- classical monocytes, on the other hand, are defined being CD14-/CD16+ with pro-inflammatory characteristics and main effector function being antigen presentation [2]. Intermediate monocytes, defined as CD14+/CD16+, create a hybrid of both pro- and anti-inflammatory characteristics such as phagocytosis and antigen presentation [2]. The current thought is that the different subpopulations of monocytes may preferentially select for a future macrophage phenotype, pro- or anti-inflammatory [2]. Therefore, understanding the role of fluid shear stress (FSS) in directing changes to monocyte subtype could predict poorer prognosis with an emergence of a certain subtype, such as increasing the intermediate monocyte population. We propose that exposure of monocytes to shear stress will increase the intermediate monocyte population and will thus exhibit both pro- and anti-inflammatory characteristics.

Materials and

Methods: : Monocytes were isolated, using a pan monocyte isolation kit, from peripheral whole blood collected from the Gulf Coast Regional Blood Center. Isolated monocytes were plated in untreated 6-well plates at a concentration of 500,000 cells/mL. After 24hr incubation, the primary monocytes were exposed to 30 min of cyclical fluid shear stress in the Integra Viaflow at 3 different accelerations at the tip of the needle: 11.63, 25.04, and 62.30 dyne/cm^2. The Integra Viaflow machine is an automatic 96 well pipettor, that controls the speed in which the cells are de-plunged from the pipette tips. Our lab has created specific pipette tips that fuse the body of a pipette with the blunt needle of a syringe in order to generate greater shear forces at the tip of the syringe, even if cells are only exposed to those forces for a few seconds [3].The sheared mono were then allowed to incubate for 24, 48, and 72 hr. Following the incubation period, cells were prepped for confocal imaging, flow cytometry, and the media frozen for ELISA assays to measure NFkB phosphorylation, key cell surface marker expression, and cytokine secretion.

Results, Conclusions, and Discussions:: Results
Monocytes that were exposed to 62.30 dyne/cm^2 were found to have significantly enhanced NFkB phosphorylation 4 hr after FSS was applied when compared to both static and other sheared groups (Fig1A,B). Interestingly, primary monocytes did not secret TNF-⍺ 24 hours following FSS exposure, however after 48 hr all 3 sheared groups secreted TNF-⍺ where the static condition did not (Fig1C). Sheared monocytes were found to have a reduction in CD206 and CD86 as shear stress increases, where in CD163 it increased for 11.63 dyne/cm^2 and returned back to static levels 48 hr post FSS (Fig1D). A similar trend that was noted in CD163 was also seen in CD16 and CD14, where 11.63 dyne/cm^2 elicited the highest intensity and all other conditions were similar (Fig1E,F). Monocytes exposed to 11.63 dyne/cm^2 as a reduction in CD80 and a return to baseline in 25.04 and 62.30 dyne/cm^2, the opposite of CD163 (Fig1G).
Discussion and Conclusions
Shear stress can affect the subpopulation of monocytes differently, where classical monocytes may be more likely to acquire anti-inflammatory M2-like characteristics such as CD206 and CD163 expression. Monocytes exposed to speed 1 for 30 min was found to have high expression of CD16, CD14, CD86, CD206, and CD163 and a low expression of CD80, along with an increase in TNF-⍺ secretion, meaning the acquisition of both pro- and anti-inflammatory characteristics. Monocytes that underwent 11.63 dyne/cm^2 seems to increase the population of intermediate monocytes. Because of the acquisition of both pro- and anti-inflammatory characteristics, these populations of monocytes may play a significant role in controlling tumor progression by being more tumor supportive. A similar phenomenon occurs in the various phenotypes of anti- inflammatory macrophages, where the subtype of macrophage, M2b, expresses both CD163 and CD86 and thus promoting a tumor supportive microenvironment. Ongoing research is addressing the effects of monocytes as it relays to cancer metastasis by co-culturing monocytes with cancer cells, since both monocytes and circulating tumor cells are circulating in the bloodstream, to understand the effects of the various monocyte subpopulations have on cancer cell survival.

Acknowledgements and/or References (Optional):: Reference
1. Patysheva, M. et al. (2022) ‘Monocyte programming by cancer therapy’, Frontiers in Immunology, 13. doi:10.3389/fimmu.2022.994319.
2. Mukherjee, R. et al. (2015) ‘Non-classical monocytes display inflammatory features: Validation in sepsis and systemic lupus erythematous’, Scientific Reports, 5(1). doi:10.1038/srep13886.
3. Fabiano, A.R. et al. (2024) ‘Multiplex, high-throughput method to study cancer and immune cell mechanotransduction’, Communications Biology, 7(1). doi:10.1038/s42003-024-06327-x.