Biophysical signatures of macrophage activation by cytokines or through Fc receptors

Introduction:: Macrophage plasticity is well appreciated but incompletely understood. At least in vitro, a spectrum of macrophage polarization states exists in response to stimulation with cytokines, pathogen- or damage-associated molecular patterns, and other immune modulatory signals. These states are typically characterized by specific surface markers and cytokine secretions, but differential gene expression among polarized macrophage subsets also indicates alterations in adhesive protein repertoires and cytoskeleton organization that are likely to influence function. In other cell types including embryonic germ layer cells and cancer cells, differential adhesion and differential contractility between cell populations results in cell sorting, but such phenomena have not been explored in immune cells including macrophages. Here, we activate mouse macrophages in vitro with various polarizing stimuli and describe their propensity to aggregate on low-adhesive materials as monocultures and in mixtures with cancer cells. We further identify macrophage aggregates in mouse tumors following treatment with a monoclonal antibody (mAb) that activates Fc receptors for phagocytosis, which suggests that our in vitro findings are potentially relevance to macrophage immunobiology in disease and therapy.

Materials and Methods:: The macrophages used in this study were differentiated from primary mouse bone marrow cells or Hoxb8-conditionally immortalized macrophage progenitors. Differentiated macrophages were detached from culture dishes, labeled with CellTracker fluorescent dyes, and replated on low adhesive surfaces with mouse interferon-γ (IFNγ) and/or lipopolysaccharide (LPS), interleukin-4 (IL-4), or the myosin-II inhibitor blebbistatin. Macrophages were imaged by fluorescence microscopy beginning the next day. In some experiments, macrophages and cytokines were added to pre-formed ‘tumoroid’ aggregates of B16 melanoma cells on the same low-adhesive surfaces along with a tumor-opsonizing monoclonal antibody (anti-Tyrp1) [1]. For in vivo assessment of macrophage clustering, tumors were produced by subcutaneous injection of B16 cells in syngeneic mice. Tumor-bearing mice were injected intravenously with anti-Tyrp1 one day prior to tumor isolation for fixation, sectioning, and immunofluorescence staining for the macrophage marker F4/80. Animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania.

Results, Conclusions, and Discussions:: Macrophages cultured on low adhesive substrates exhibit diverse aggregation behavior when activated by different polarizing stimuli. Specifically, pro-inflammatory activation with IFNγ, but not LPS, promotes aggregation of macrophages into tight clusters while activation with IL-4 drives macrophage dispersion (Figure 1A). Inhibiting myosin-II-based cell contractility also favors macrophage clustering. This finding seems consistent with transcriptomics of IFNγ-treated mouse macrophages showing decreased expression of genes associated with contractility as well as changes in adhesion receptor expression. In co-culture aggregates of mouse macrophages and mouse B16 melanoma cells, macrophages exhibit a striking clustering when activated by IFNγ (Figure 1B). Clustering is also observed among macrophages phagocytosing B16 cells opsonized with a monoclonal IgG antibody, although the aggregation in this case is transient and macrophages disperse after eliminating B16. Importantly, macrophage aggregates are also observed in B16 tumors in mice treated systemically with the opsonizing antibody [1].

Our results reveal a potential new biophysical signature of macrophage polarization, namely the propensity of different subsets of macrophages to aggregate due to favorable homotypic cell-cell interactions or to disperse due to less favorable homotypic interactions compared to cell-substrate or heterotypic cell-cell interactions. The molecular pathways underlying this phenomenon likely include changes to cytoskeletal organization and adhesion receptor expression upon polarization, although the specific details remain to be elucidated. These new insights should be broadly applicable to macrophage immunobiology in inflammation and cancer and especially to macrophage-based cancer immunotherapy.


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References (Optional): : [1] Dooling, L.J., Andrechak, J.C. et al. Cooperative phagocytosis of solid tumours by macrophages triggers durable anti-tumour responses. Nat. Biomed. Eng (2023). https://doi.org/10.1038/s41551-023-01031-3