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AFM-IR Probes the Influence of Polarization on the Expression of Proteins within Single Macrophages

Update time:2021-06-15
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Macrophages, as primary cells of the innate immune system, contribute in a variety of biological processes, such as host defense and wound healing. To fulfill their typical functional roles, macrophages are polarized into a spectrum of phenotypes depending on distinctive signals, such as classical (proinflammatory, M1) and alternative (anti-inflammatory, prohealing, M2) activation states.


By adjusting the polarization of macrophages, different phenotypes of macrophages play a key role in the prognosis and outcome of the disease. It is thought that the application of an inducer in the extracellular environment is an effective means of manipulating the polarization process of macrophages, however the lack of effective characterization methods makes it difficult to reveal the microscopic mechanism of a series of biological processes and the effect of inducer on the biochemical properties within single microphages.


Recently,the research group at Shanghai Institute of Ceramics of the Chinese Academy of Sciences in cooperation with Fudan University, used advanced atomic force microscopy to in-situ characterize the effects of IL-13 and LPS inducers on the cytokines and secreted within single microphages macrophages on the nanoscale. the nanoIR spectrum reveals that the content of the secondary structure in the elongated part of M1/M2 displays significant differences. M1 mainly contains 35% antiparallel β-sheets (due to high expression of TNF-α), while M2 is made up of 39% α-helices. The difference in secondary structure stems from the difference in the concentration and type of expressed protein after different polarizations. M1 expresses high levels of TNF-α and IL-6 cytokines, while M2 expresses high levels of IL-4 and IL-10 cytokines. The ultra-high resolution nanochemical image shows the nano-scale spatial distribution of characteristic proteins in a single M1/M2 phenotype polarized macrophage, and the high-throughput nano-elastic in-situ quantitative characterization further reveals the nano-scale biomechanics of polarized macrophages. These new characterization results will provide new enlightenment and new ideas for understanding the correlation between macrophages' nanoscale structure, biological functions and physical properties. The result was published in J. Mater. Chem. B, 9 (2021) 2909–2917.


The research work is supported by the National Key Research and Development Program and the instrument developing program of Chinese Academy of Sciences.


Fig. 1 Tapping-IR biochemical mapping of polarized macrophages. (a, f) Topography images and infrared absorption mappings at (b and g) 1550 cm-1, (c and h) 1652 cm-1, (d and i) 1687 cm-1, and (e and j) 1710 cm-1, and high-resolution tapping-IR mapping of the elongated part of M1 (k) and M2 (l).


Fig. 2 Secondary structure content determinated from nanoIR absorption spectra collected at the elongated part of polarized macrophages. Morphologies and nano-IR spectra of (a) M1 and (d) M2 macrophages (scale bar: 10 μm). (b and e) Deconvolution of the structural contributions in amide band I by second-derivative and fourth-derivative analysis. (c and f) The averaged spectrum and deconvolution fitting of the spectrum with Gaussian–Lorentzian functions. (g) Comparison of the measured secondary structure content in M1/M2 macrophages.



Prof. ZENG Huarong

Shanghai Institute of Ceramics