Poster V13 - Immuno-Mechanical Regulation of Myeloid Cells

Introduction: Myelofibrosis is a progressive bone marrow disease associated with aberrant immune cells and inflammation, resulting in scarring of the bone marrow. Monocytes, a type of myeloid cell, are one of the several immune cells implicated in the pathological remodeling of the extracellular matrix, as it has been demonstrated that monocyte-derived fibrocytes contribute to bone marrow fibrosis induction in primary myelofibrosis (Verstovsek et al., 2016). Previous work has also shown that altering the mechanical properties of the monocyte environment can modulate their immunogenicity and differentiation (Vining et al., 2022).
To further investigate the immunomodulatory effects of viscoelasticity and stiffness in the context of myelofibrosis, myeloid cells were encapsulated in a tunable artificial extracellular matrix (aECM) hydrogel system. The aECM system employed permits the tuning of viscoelasticity and stiffness, while keeping the hydrogel’s microscale structure consistent (Vining et al., 2019). Cell surface markers before and after encapsulation, as well as pro-inflammatory cytokine, IL1- Β and Il-6, secretion were quantified to characterize the identity and immunogenicity of myeloid cells in aECM of differing stiffness and viscoelasticity.

Materials and

Methods: Myeloid cells were generated by differentiation from mobilized enriched CD34+ HSPC cells from the Fred Hutch Cancer Center. A CD34+ HSPC cell bank was generated by culturing the cells for 7-8 days in serum-free StemSpan medium (Stem Cell Technologies) supplemented with stem cell factor, Flt-3 ligand, TPO, IL-6, IL-3, and UM729. For differentiation, HSPCs were cultured in IMDM supplemented with GM-CSF and Flt-3 ligand for 7-8 days. One day prior to encapsulation, myeloid cells are stimulated using IMDM supplemented with GM-CSF and IL-4.

The aECM used in this work comprises 1.5 wt% very low viscosity alginate, 4 mg/ml collagen type I, varying concentration of calcium carbonate particles, and HBSS buffer with HEPES and NaOH. The Ca ions are released from the particles through the addition of glucono-delta-lactone (GDL), a weak acid which has a pH that drops steadily over time. This mechanism enables the alginate to crosslink while the collagen matrix self-assembles at 37°C. Cells were encapsulated at a density of 5 million cells per ml of gel solution.

The mechanical properties of the aECM are assessed by the Discovery HR30 rheometer under the oscillatory mode. The gel solution was subjected to 2% strain at 0.1 Hz under a time sweep until the storage and loss modulus equilibrated at 37°C. The effective elastic modulus was estimated by assuming a 0.5 Poisson’s ratio.

Results, Conclusions, and Discussions: Our rheology data characterizes the aECM hydrogel system used for encapsulation. These data show that we were able to generate four aECM conditions of varying stiffness and viscoelasticity (Fig. 1). Our flow cytometry results demonstrate that after encapsulation, the CD14+ population is reduced across all conditions, suggesting that the classical monocyte population is lost after encapsulation (Fig. 2). We further probed for dendritic cell (DC) surface markers. After encapsulation, there is an increase in the CD11c+ population, suggesting the myeloid cells are differentiating down a DC lineage. Further gating on the CD45+ HLADR+ CD14- CD11c+ population with CD1c and CD16 shows that the most stiff, elastic gel condition yields the lowest CD16+ population, while the CD1c population remains similar across conditions (Fig. 3). Studies have shown that the CD16+ DC population is more migratory compared to the CD16- subtypes and they reverse transmigrate to activate T-cell proliferation. Therefore, our flow cytometry results suggest that tissues that are stiff and elastic suppress the migratory population of DCs (Villani et al., 2017, Randolph et al., 2002).

Quantification of IL1-β secretion by ELISA suggests that IL1-β secretion is increased when myeloid cells are encapsulated in a softer and more viscous aECM (p < 0.01) (Fig. 4). There was no significant difference in IL1-β secretion at different stiffnesses for myeloid cells within the more elastic aECM. ELISA-based quantification also show that with IFN-ɣ stimulation, myeloid cell IL-6 release is not significantly affected by aECM stiffness and viscoelasticity (Fig. 4).
This work sheds light on the effect of stiffness and viscoelasticity on the immunogenic profile of myeloid cells. To identify key regulatory pathways and potential gene targets for myelofibrosis, future work may involve investigating the cytoskeletal organization, epigenetic changes, and gene expression changes of myeloid cells in different aECM.

Acknowledgements (Optional): Acknowledgements:
NSF GRFP
NIH/NIDCR R00DE030084 Research Scholar Grant
American Cancer Society (RSG-23-1152051-01-MM) Pilot Seed Grant
Center for Engineering MechanoBiology (NSF Grant CMMI 15-48571) Pilot Grant
Penn Center for Musculoskeletal Disease (NIH/NIAMS P30AR069619)

References:
Verstovsek, S., Manshouri, T., Pilling, D., Bueso-Ramos, C. E., Newberry, K. J., Prijic, S., Knez, L., Bozinovic, K., Harris, D. M., Spaeth, E. L., Post, S. M., Multani, A. S., Rampal, R. K., Ahn, J., Levine, R. L., Creighton, C. J., Kantarjian, H. M., & Estrov, Z. (2016). Role of neoplastic monocyte-derived fibrocytes in primary myelofibrosis. Journal of Experimental Medicine, 213(9), 1723–1740. https://doi.org/10.1084/jem.20160283

Vining, K. H., Marneth, A. E., Adu-Berchie, K., Grolman, J. M., Tringides, C. M., Liu, Y., Wong, W. J., Pozdnyakova, O., Severgnini, M., Stafford, A., Duda, G. N., Hodi, F. S., Mullally, A., Wucherpfennig, K. W., & Mooney, D. J. (2022). Mechanical checkpoint regulates monocyte differentiation in fibrotic niches. Nature Materials, 21(8), 939–950. https://doi.org/10.1038/s41563-022-01293-3

Vining, K. H., Stafford, A., & Mooney, D. J. (2019). Sequential modes of crosslinking tune viscoelasticity of cell-instructive hydrogels. Biomaterials, 188, 187–197. https://doi.org/10.1016/j.biomaterials.2018.10.013

Villani, A.-C., Satija, R., Reynolds, G., Sarkizova, S., Shekhar, K., Fletcher, J., Griesbeck, M., Butler, A., Zheng, S., Lazo, S., Jardine, L., Dixon, D., Stephenson, E., Nilsson, E., Grundberg, I., McDonald, D., Filby, A., Li, W., De Jager, P. L., … Hacohen, N. (2017). Single-cell RNA-seq reveals new types of human blood dendritic cells, monocytes, and progenitors. Science, 356(6335). https://doi.org/10.1126/science.aah4573

Randolph, G. J., Sanchez-Schmitz, G., Liebman, R. M., & Schäkel, K. (2002). The CD16+ (FCΓRIII+) subset of human monocytes preferentially becomes migratory dendritic cells in a model tissue setting. The Journal of Experimental Medicine, 196(4), 517–527. https://doi.org/10.1084/jem.20011608