(E-197) Fluid Shear Stress Mechanotransduction Enhances Dendritic Cell Activation

Introduction:: Cancer therapeutics have been developed in which a patient’s immune cells are activated ex vivo and reintroduced into the body for lymph node homing and T cell priming [1]. As some of these therapeutics only increase life expectancy by a few months, finding a way to increase activation ex vivo is essential [1]. Cells can be activated by shear stress via mechanosensitive ion channels opening in response to a mechanical stimulus and initiating mechanotransduction via calcium influx [2]. In dendritic cells (DCs), these responses include growth and proliferation [3]. Cone-and-plate and other flow devices can be used to apply fluid shear stress (FSS) to cells, and the structure of the apparatus ensures that all cells experience the same level of FSS [4]. Here, we investigated the resulting effects on DCs with applied FSS stimulation.


Materials and Methods:: DC2.4 murine dendritic cells were grown in culture and suspended in complete RPMI media. Primary human DCs were isolated from whole blood according to the manufacturer’s instructions (Miltenyi Biotec) and primary mouse bone marrow derived dendritic cells (BMDCs) were isolated as described in the literature [5]. FSS of 5 dyn/cm² was applied via a cone-and-plate flow device to DCs for 1 h, and compared to control conditions at 0 dyn/cm². Cells were plated overnight or immediately fixed, permeabilized and stained with antibodies for flow cytometry analysis. NF-𝜅B and c-Fos phosphorylation, RAC1 expression, proliferation and pH were analyzed after 1 h of FSS. After 24 h, costimulatory molecules, cytokines and chemokines were analyzed using flow cytometry and Proteome Profiler Assay. Imaging analyses were performed via confocal microscopy.


Results, Conclusions, and Discussions:: Results and Discussion: After 1h FSS stimulation, there were significant changes in metabolism such as enhanced glucose uptake and pH level, as measured by pH meter (Fig 1A). Similarly, morphologic changes, which are indicative of DC activation, occurred following FSS stimulation, with an increase in dendrite formation and an increase in RAC1 expression in shear conditions (Fig 1B-C). Proteome profiler assay revealed significant increased production of multiple cytokines following FSS, including TNF-a, ICAM-1, CCL3, and CXCL2 (Fig 1E-F). NF-kB and c-Fos phosphorylation were increased following FSS stimulation in primary mouse BMDCs (Fig 1G-H).

Conclusions: After just 1h of applied FSS, DCs showed significant changes in metabolism, morphology, and cytokine release, indicating their activation. Increased expression of transcription factors NF-𝜅B and AP-1 gives insight to pathways involved in DC activation via FSS. The results suggest that FSS could be used in DC-based ex vivo immunotherapies to enhance efficacy.


Acknowledgements (Optional): : This work was supported by NIH Grant R01 CA203991 and the NSFGRFP.

References (Optional): : [1] E Anassi, P T, 2011, 36, 197-202; [1] JM Hope, Cell Death & Dis, 2019, 10, 1-15; [2] MJ Mitchell, PNAS, 2014, 111, 930-935; [4] E Shumilina, Am J Physiol Cell Physiol, 2011, 300, C1205-1214; [3] JS Lewis, Biomaterials, 2014, 34, 9063-9070; [5] A Madaan, JBM, 2014, 1, 1.