Mechanosensing regulates tissue repair program in macrophages

Tissue-resident macrophages play important roles in tissue homeostasis and repair. However, how macrophages monitor and maintain tissue integrity is not well understood. The extracellular matrix (ECM) is a key structural and organizational component of all tissues. Here, we find that macrophages sense the mechanical properties of the ECM to regulate a specific tissue repair program. We show that macrophage mechanosensing is mediated by cytoskeletal remodeling and can be performed in three-dimensional environments through a noncanonical, integrin-independent mechanism analogous to amoeboid migration. We find that these cytoskeletal dynamics also integrate biochemical signaling by colony-stimulating factor 1 and ultimately regulate chromatin accessibility to control the mechanosensitive gene expression program. This study identifies an “amoeboid” mode of ECM mechanosensing through which macrophages may regulate tissue repair and fibrosis.

The PDF file includes: Figs. S1 to S5 Table S1 Legend for movies S1 to S4 Other Supplementary Material for this manuscript includes the following:

Rpl13a
Forward GAAGGAAAAGGCCAAGATGCAC Reverse TGAGGACCTCTGTGAACTTGC Table S1.List of primers used for qPCR analysis

Fig. S1 .
Fig. S1.Mechanical properties of collagen gels and PA hydrogels (A) Stiffness of low-and high-collagen gels, measured by rheology.(B) Scanning electron microscopy (SEM) images of low-and high-collagen gels at lower (above) and higher (below) magnification.(C) Pore sizes of low-and high-collagen gels quantified in SEM images.(D) Relative gene expression in BMDMs cultured in indicated concentration of collagen gels with or without IL-4.(E) Gating strategy for flow cytometry analysis of FIZZ1 and ARG1 protein expression in BMDM.(F) Stiffness of low-and high-stiffness PA gels, measured by rheology.Data are represented as mean ± SD.Student's t test (A, B, and F) and two-way ANOVA (D) are used for statistical analysis.*** p < 0.001, **** p < 0.0001.ns; not significant.

Fig. S2 .
Fig. S2.Fibroblast mechanosensing is integrin-and non-muscle myosin II-dependent.(A) Relative gene expression in MEFs cultured in low-or high-collagen gels with the indicated doses of blebbistatin or DMSO (equivalent to the amount of DMSO in the lowest and highest concentrations of blebbistatin).(B) Relative gene expression in MEFs cultured in 3D lowcollagen gels with β1 integrin-blocking antibody or isotype control antibody.Data are represented as mean ± SD.Two-way ANOVA (A) and Student's t test (B) were used for statistical analysis.*p < 0.05, ** p < 0.01, **** p < 0.0001.ns; not significant.

Fig. S3 .
Fig. S3.Macrophages show minimal expression of collagen-binding integrins.(A)Normalized gene expression levels (transcripts per million, TPM) of all integrin alpha and beta chains in BMDMs, from RNA-seq analysis of BMDMs cultured in 3D low-or highcollagen gels, with or without IL-4 stimulation.(B) Normalized gene expression levels (TPM) of all integrin alpha and beta chains in various tissue-resident macrophage populations, based on reanalysis of RNA-seq data from Lavin et al.56 Alpha chains of collagen-binding integrins are highlighted in green (Itga1, Itga2, Itga10, Itga11).Data are represented as mean ± SD.

Fig
Fig. S4.YAP signaling and mechanosensitive ion channels are not required for macrophage mechanosensing.(A) TPM of Yap1, Piezo1 and Trpv4 from RNA-seq analysis of BMDMs cultured in 3D low-or high-collagen gels with or without IL-4 stimulation.(B) Relative gene expression in BMDMs treated with control or K-975 (TEAD inhibitor) and cultured in low-or high-collagen gels in the presence of IL-4.(C) Relative gene expression in BMDMs derived from Piezo1 f/f or LysM Cre/+ Piezo1 f/f mice, cultured in low-or high-collagen gels in the presence of IL-4.(D) Relative gene expression in BMDMs treated with control, RN-1734 or HC-067047 (TRPV4 inhibitors), or GsMTx4 (mechanosensitive ion channel inhibitor) and cultured in low-or high-collagen gels in the presence of IL-4.Data are represented as mean ± SD.Two-way ANOVA was used for statistical analysis.*p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Fig. S5 .
Fig. S5.Mechanosensitive gene expression program is induced during tissue repair in vivo.(A) Gating strategy for flow cytometry analysis of FIZZ1 and ARG1 protein expression in mouse lung macrophages.(B) FIZZ1 and ARG1 protein expression in lung macrophages in PBSor bleomycin-treated mice, analyzed by flow cytometry.Representative plots of CD64 + F4/80 + cells following the gating strategy in (A) are shown.