(WHS-K2.02) DIRECT CONTACT WITH MECHANICALLY ACTIVATED MYOFIBROBLASTS DRIVES MACROPHAGES INTO DISTINCT TRANSCRIPTIONAL AND FUNCTIONAL STATES
Thursday, May 16, 2024
9:15 AM – 10:15 AM East Coast USA Time
Background: Both fibroblasts and macrophages (MΦ) are key in promoting the formation and remodeling of extracellular matrix following organ injury, but aberrant crosstalk can contribute to the development of fibrosis. MΦ provide cytokines like TGF-β1 that stimulate fibroblast activation into contractile myofibroblasts (MF). We have published that MF activation by MΦ-derived TGF-β1 requires spatial proximity and a 'scar-stiff' tissue environment. However, little is known how, in turn, MFs control MΦ phenotypes in the context of tissue repair and fibrosis. We hypothesized that mechanically activated MFs control distinct MΦ states in contact–dependent signaling processes.
Methods: MΦ were obtained by treating mouse bone marrow-derived monocytes with M-CSF in vitro for 5 d. Subcutaneous fibroblasts were isolated from Col1a-GFP reporter mice. To mechanically establish fibroblast and MF populations, fibroblasts were cultured on skin-soft or scar-stiff gelatin-coated silicone substrates for 2 passages, respectively. MΦ were then co-cultured for 3 d with fibroblasts/MFs on the respective substrates in setups that allowed either direct contact or medium-sharing only. Cells obtained from all experimental combinations were separately analyzed using immunofluorescence (IF) confocal microscopy and flow cytometry. Fibroblastic cells and MΦ were flow-sorted for subsequent RNA sequencing, further analyzed for principal components, differentially expressed genes and enrichment of signaling pathways and transcription factors binding motifs.
Results: Fibroblasts cultured alone on stiff substrates exhibit MF protein and RNA profiles absent from soft-cultured fibroblasts. Substrate stiffness in the chosen range does not affect RNA profiles of MΦ in monoculture. Conversely, co-culture with fibroblastic cells results in significant changes in MΦ transcriptomes, with unique features depending on the activation state of the co-cultured fibroblasts and the ability to form direct contact. Specifically, (1) MΦ in direct but not medium-shared-only co-culture with fibroblastic cells acquire an activated MAPK signaling. (2) Direct contact with fibroblasts results in suppression of stress response to stimuli and inter- and intra-cellular signal transduction. (3) In direct contact with MFs, MΦ exhibit upregulated pro-fibrotic signaling pathways, mediated by the activation of sema-plexin-GTPase axis and IL-17, NF-kB, and C-type lectin signaling. IF and flow cytometry validate RNA sequencing data. For instance, MΦ exhibit up to 1.6-fold significantly increased expression of CD206 in MΦ co-cultured with MFs versus fibroblasts. Increased expression of MΦ CD206 is 6~8-fold more pronounced in co-cultures with direct contact.
Conclusion: Direct contact with MFs generate a unique MΦ polarization state. The recognition of such a state offers novel therapeutic targets and potential for the prevention and treatment of fibrosis.