Direct recognition of invading pathogens by innate immune cells is a critical driver of the inflammatory response. However, cells of the innate immune system can also sense their local microenvironment and respond to physiological fluctuations in temperature, pH, oxygen and nutrient availability, which are altered during inflammation. Although cells of the immune system experience force and pressure throughout their life cycle, little is known about how these mechanical processes regulate the immune response. Here we show that cyclical hydrostatic pressure, similar to that experienced by immune cells in the lung, initiates an inflammatory response via the mechanically activated ion channel PIEZO1. Mice lacking PIEZO1 in innate immune cells showed ablated pulmonary inflammation in the context of bacterial infection or fibrotic autoinflammation. Our results reveal an environmental sensory axis that stimulates innate immune cells to mount an inflammatory response, and demonstrate a physiological role for PIEZO1 and mechanosensation in immunity.
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We thank J. Alderman, C. Lieber, C. Hughes, E. H.-P. and P. Rainey for help in facilitating this work; B. Kazmierczak for providing the P. aeruginosa; M. Roulis for insightful comments and reagents; L. Orr for helpful insight regarding statistical analysis; I. Odell for help with computational software; C. Rothlin and Dr. C. Abraham for continued support and feedback as this work progressed; P.-M. Chen for help and reagents related to hypoxia studies. This work was supported by the Howard Hughes Medical Institute and the Blavatnik Family Foundation (R.A.F.). This work was supported in part by the Searle Scholars Program, the Leukemia Research Foundation, the Gruber Foundation and the NIH (R01GM122984). R.J. was supported in part by the Crohn’s and Colitis Foundation. M.A.S. was supported by the NIH (HL RO1 75092). A.G.S. was supported in part by an NIH training grant (T32 GM007499) and the American Society for Microbiology.
: R.A.F. is a scientific advisor to GlaxoSmithKline, a consultant for Hatteras Venture Partners and a shareholder and consultant for Zai Lab Ltd. All other authors declare no competing interests.
Peer review information Nature thanks Seth Alper, Luke O’Neill, Sarah Walmsley and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
a, Schematic of CHP bioreactor showing side view. b, Graph of pressure regimes used within the pressure chamber. c, RT–qPCR analysis of Piezo1 in unstimulated BMDMs from Piezo1fl/fl (WT) and Piezo1ΔLysM mice. Data are presented as five biological replicates from two independent experiments. d, RT–qPCR analysis of Piezo1fl/fl BMDMs treated with static pressure at the indicated magnitude or with CHP for 6 h. Data are presented as four biological replicates from two independent experiments. e, Immunoblot analysis of HIF1α and β-tubulin from Piezo1fl/fl BMDMs treated with static pressure at the indicated magnitude or with CHP for 6 h. Data are representative of two independent experiments. f, ELISA of IL-1β from Piezo1fl/fl and Piezo1ΔLysM BMDMs treated with CHP alone (NT), 10 ng ml−1 LPS with 5 h CHP followed by 1 h CHP, 5 h CHP followed by 1 h nigericin (10 μM) in CHP, or 10 ng ml−1 LPS for 5 h followed by 1 h nigericin (10 μM) with CHP. Data are presented as three biological replicates. Data in c, d, f are mean ± s.e.m.; significance is determined by unpaired two-tailed t-test (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001).
a, Representative confocal microscopy of BMDMs treated for 6 h in either normoxic conditions or in a hypoxic chamber (2% O2). Cells were treated with pimonidazole (20 μM) for 1 h before completion of treatment. Upon completion, cells were fixed, permeabilized and stained with monoclonal antibody against pimonidazole. Data are representative of two independent experiments. b, Confocal microscopy of BMDMs treated for 6 h in either static, CHP or hypoxic chamber (0.5% O2). Cells were treated with 20 μM pimonidazole 1 h before completion of treatment. Upon completion, cells were fixed, permeabilized and stained with monoclonal antibody against pimonidazole. Data are representative of two independent experiments. c, RT–qPCR analysis of BMDMs treated with cyclical hydrostatic pressure, 2% O2 or cyclical hydrostatic pressure with 2% O2 for 6 h. Data are presented as three biological replicates. d, RT–qPCR analysis of Hif1a in BMDMs following CHP. Data are presented as four biological replicates. Data are from two independent experiments. e, Representative immunoblot analysis of HIF2α and β-tubulin from BMDMs treated with static pressure, CHP or DMOG (200 μM) for 6 h. Data in c, d are mean ± s.e.m.; significance is determined by unpaired two-tailed t-test.
Extended Data Fig. 3 Cyclical hydrostatic pressure-induced HIF1α protein is stabilized independent of acidity.
a, Intracellular pH measurements using pHrodo intracellular pH indicator dye for final 30 min of treatment. Piezo1fl/fl (n = 3) and Piezo1ΔLysM (n = 3) cells were treated with CHP for the indicated periods or with low-pH cell medium for 6 h. b, pH measurements of supernatant from Piezo1fl/fl (n = 3) and Piezo1ΔLysM (n = 3) BMDM culture treated with CHP for the indicated periods. Data in a, b, are mean ± s.e.m.; significance is determined by unpaired two-tailed t-test.
Extended Data Fig. 4 Kinetics and sufficiency of cyclical hydrostatic pressure induced HIF1α stabilization and transcriptional response.
a, Representative immunoblot analysis of HIF1α and β-tubulin from BMDMs in CHP with 5 μM GsMTx4 (Gsm) for 6 h, then washed three times in DMEM before further CHP stimulation. Data are representative of two independent experiments. b, Immunoblot analysis of HIF1α and β-tubulin from BMDMs in CHP for 6 h, pretreated with 10 μM BAPTA-AM for 30 min. Data are representative of two independent experiments. c, Immunoblot analysis of HIF1α and β-tubulin in BMDMs. Piezo1fl/fl (WT) or Piezo1ΔLysM (KO) were cultured in CHP for 6 h along with 3 μg ml BFA. Cells were then washed with DMEM three times and further subjected to additional CHP stimulation. Supernatant (sup) was then clarified and transferred to WT or KO BMDMs cultured in static pressure for two additional hours. Data are representative of two independent experiments. d, Representative immunoblot analysis of HIF1α and β-tubulin from BMDMs treated with 100 μM chloroquine (ChlQ) or 50 μM MG132 for 6 h with CHP. Data are representative of two independent experiments. e, RT–qPCR analysis of Edn1 from Hif1afl/fl and Piezo1ΔLysM BMDMs treated with CHP for 1 h or 6 h. Data are presented as three biological replicates. f, g, RT–qPCR analysis of Piezo1fl/fl (f) or Piezo1ΔLysM (g) BMDMs cultured in 6 h of treatment 200 μM DMOG, 6 h of CHP, 2 h of CHP followed by 4 h of no treatment (NT), 2 h of static pressure followed by 4 h of 200 μM DMOG, or 2 h CHP followed by 4 h of 200 μM DMOG. Data are representative of three biological replicates. Data are mean ± s.e.m.
a–c, RT–qPCR analysis of known mammalian mechanosensory ion channels from monocytes (a), alveolar macrophages (b) or neutrophils (c) sorted from lungs of mice infected with P. aeruginosa or PBS for 6 h. Data are represented as fold increase over neutrophil expression for each gene. Data are representative of four biological replicates. Data are mean ± s.e.m.
a, Representative flow cytometry of lungs from Piezo1fl/fl (top; n = 9) or Piezo1ΔLysM (bottom; n = 9) mice infected intranasally with P. aeruginosa for 6 h. The lymphoid lineage is TCRβ+NK1.1+B220+CD19+CD90.2+TCRγδ+. Data are representative of two independent experiments. b, c, Infiltrating monocyte quantification in terms of percentage(b) and total numbers (c) following 6 h of intranasal P. aeruginosa infection of Piezo1fl/fl (n = 7) or Piezo1ΔLysM (n = 7) mice. Data are representative of two independent experiments. d, Representative intracellular flow cytometry histograms of HIF1α from total lung interstitial monocytes of Piezo1fl/fl and Piezo1ΔLysM mice infected intranasally with P. aeruginosa for 6 h. Data are from two independent experiments. e, MFI of HIF1α from in vivo-labelled CD45.2+ circulatory monocytes from Piezo1fl/fl (n = 7) or Piezo1ΔLysM (n = 6) mice infected with P. aeruginosa for 6 h. Data are from two independent experiments. f, MFI of HIF1α from tissue-infiltrated and circulatory monocytes from mice infected with P aeruginosa intranasally (n = 7) or intraperitoneally (n = 8) for 6 h. Data are from two independent experiments. g, RT–qPCR analysis from BMDMs treated with oscillatory shear stress for 6 h. Data are representative of three biological replicates. Data in b, c, e–g are mean ± s.e.m.; significance is determined by unpaired two-tailed t-test (****P < 0.0001).
a, CFUs from liver of Piezo1fl/fl mice infected intranasally with P. aeruginosa for 24 h, pre-treated with 25 μg EDN1-blocking antibody (n = 5) or isotype control (n = 6) intranasally 6 h before infection. Data are from two independent experiments. b, CFUs from liver of Piezo1ΔLysM mice infected intranasally with P. aeruginosa for 24 h treated with vehicle (n = 10) or 10 μg rEDN1 (n = 9) at the time of infection. Data are from two independent experiments. c, d, CFUs from lung(c) or liver(d) of Piezo1fl/fl mice infected intranasally with P. aeruginosa for 24 h, pre-treated with vehicle (n = 10) or 100 mg per kg (body weight) bosentan (n = 8) intraperitoneally 3 h before infection. Data are from two independent experiments. Data are mean ± s.e.m.; significance is determined by unpaired two-tailed Mann–Whitney U-test (*P < 0.05,**P < 0.01 and ***P < 0.001).
Cyclical force signals via PIEZO1 in myeloid cells, resulting in Ca2+ influx and AP-1-induced EDN1 expression. Ednrb signalling then drives HIF1α stabilization and proinflammatory transcriptional upregulation.
Extended Data Fig. 9 Infiltrating monocytes recognize cyclical hydrostatic pressure in the lung via PIEZO1 to trigger neutrophil-mediated bacterial clearance.
Recruited monocytes recognize cyclical force via PIEZO1 in the lung and secrete EDN1 to drive HIF1α stabilization and CXCL2 expression to induce neutrophilia and bacterial clearance.
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Science Signaling (2019)
Nature Reviews Immunology (2019)
Arteriosclerosis, Thrombosis, and Vascular Biology (2019)