Abstract
Monocytes play essential roles in the inflammatory and anti-inflammatory processes that take place during an immune response, acting both within the vascular network and interstitially. Monocytes are activated, mobilized, and recruited in response to an inflammatory stimulus or different forms of tissue injury. The recruitment of circulating monocytes to the inflamed tissue is essential to resolving the injury.
Monocyte recruitment is a multistep process that begins with a decrease in rolling velocity, is followed by adhesion to the endothelium and crawling over the luminal vessel surface, and culminates in monocyte transmigration into the surrounding tissue. Intravital microscopy is a powerful visualization tool for the study of leukocyte behavior and function, intercellular interactions, cell trafficking, and recruitment in pathological and physiological conditions. This modality is therefore widely used for the detailed analysis of the immune response to multiple insults and the molecular mechanisms underlying monocyte interactions within the vascular system in vivo. This chapter describes a protocol for the use of intravital microscopy to analyze monocyte recruitment from the blood vessel to the inflammatory site.
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References
Libby P (2015) Fanning the flames: inflammation in cardiovascular diseases. Cardiovasc Res 107:307–309. https://doi.org/10.1093/cvr/cvv188
Leick M, Azcutia V, Newton G, Luscinskas FW (2014) Leukocyte recruitment in inflammation: basic concepts and new mechanistic insights based on new models and microscopic imaging technologies. Cell Tissue Res 355:647–656. https://doi.org/10.1007/s00441-014-1809-9
Sun H, Huo Y, Fan Z (2021) Editorial: imaging and mechanism of leukocyte recruitment and function in inflammation and infections. Front Cell Dev Biol 9:690003. https://doi.org/10.3389/fcell.2021.690003
Guilliams M, Mildner A, Yona S (2018) Developmental and functional heterogeneity of monocytes. Immunity 49:595–613. https://doi.org/10.1016/j.immuni.2018.10.005
Herrero-Fernandez B, Gomez-Bris R, Somovilla-Crespo B, Gonzalez-Granado JM (2019) Immunobiology of atherosclerosis: a complex net of interactions. Int J Mol Sci 20:E5293. https://doi.org/10.3390/ijms20215293
Braunersreuther V, Mach F (2006) Leukocyte recruitment in atherosclerosis: potential targets for therapeutic approaches? Cell Mol Life Sci 63:2079–2088. https://doi.org/10.1007/s00018-006-6127-2
Nourshargh S, Alon R (2014) Leukocyte migration into inflamed tissues. Immunity 41:694–707. https://doi.org/10.1016/j.immuni.2014.10.008
Jakubzick CV, Randolph GJ, Henson PM (2017) Monocyte differentiation and antigen-presenting functions. Nat Rev Immunol 17:349–362. https://doi.org/10.1038/nri.2017.28
Teh YC, Ding JL, Ng LG, Chong SZ (2019) Capturing the fantastic voyage of monocytes through time and space. Front Immunol 10:834. https://doi.org/10.3389/fimmu.2019.00834
Buscher K, Marcovecchio P, Hedrick CC, Ley K (2017) Patrolling mechanics of non-classical monocytes in vascular inflammation. Front Cardiovasc Med 4:80. https://doi.org/10.3389/fcvm.2017.00080
Dawson CA, Mueller SN, Lindeman GJ et al (2021) Intravital microscopy of dynamic single-cell behavior in mouse mammary tissue. Nat Protoc 16:1907–1935. https://doi.org/10.1038/s41596-020-00473-2
González-Ramos S, Paz-García M, Rius C et al (2019) Endothelial NOD1 directs myeloid cell recruitment in atherosclerosis through VCAM-1. FASEB J 33:3912–3921. https://doi.org/10.1096/fj.201801231RR
Nicolete R, Rius C, Piqueras L et al (2008) Leukotriene B4-loaded microspheres: a new therapeutic strategy to modulate cell activation. BMC Immunol 9:36. https://doi.org/10.1186/1471-2172-9-36
Rius C, Piqueras L, González-Navarro H et al (2013) Arterial and venous endothelia display differential functional fractalkine (CX3CL1) expression by angiotensin-II. Arterioscler Thromb Vasc Biol 33:96–104. https://doi.org/10.1161/ATVBAHA.112.254870
Abu-Taha M, Rius C, Hermenegildo C et al (2009) Menopause and ovariectomy cause a low grade of systemic inflammation that may be prevented by chronic treatment with low doses of estrogen or losartan. J Immunol 183:1393–1402. https://doi.org/10.4049/jimmunol.0803157
Auffray C, Fogg D, Garfa M et al (2007) Monitoring of blood vessels and tissues by a population of monocytes with patrolling behavior. Science 317:666–670. https://doi.org/10.1126/science.1142883
Clemente C, Rius C, Alonso-Herranz L et al (2018) MT4-MMP deficiency increases patrolling monocyte recruitment to early lesions and accelerates atherosclerosis. Nat Commun 9:910. https://doi.org/10.1038/s41467-018-03351-4
Sumagin R, Prizant H, Lomakina E et al (2010) LFA-1 and mac-1 define characteristically different intralumenal crawling and emigration patterns for monocytes and neutrophils in situ. J Immunol 185:7057–7066. https://doi.org/10.4049/jimmunol.1001638
Gerhardt T, Ley K (2015) Monocyte trafficking across the vessel wall. Cardiovasc Res 107:321–330. https://doi.org/10.1093/cvr/cvv147
Rius C, Sanz MJ (2015) Intravital microscopy in the cremaster muscle microcirculation for endothelial dysfunction studies. Methods Mol Biol 1339:357–366. https://doi.org/10.1007/978-1-4939-2929-0_26
Marques P, Collado A, Escudero P et al (2017) Cigarette smoke increases endothelial CXCL16-leukocyte CXCR6 adhesion in vitro and in vivo. Potential consequences in chronic obstructive pulmonary disease. Front Immunol 8:1766. https://doi.org/10.3389/fimmu.2017.01766
Molina-Sánchez P, Chèvre R, Rius C et al (2015) Loss of p27 phosphorylation at Ser10 accelerates early atherogenesis by promoting leukocyte recruitment via RhoA/ROCK. J Mol Cell Cardiol 84:84–94. https://doi.org/10.1016/j.yjmcc.2015.04.013
Chrobok NL, Jaouen A, Fenrich KK et al (2017) Monocyte behaviour and tissue transglutaminase expression during experimental autoimmune encephalomyelitis in transgenic CX3CR1gfp/gfp mice. Amino Acids 49:643–658. https://doi.org/10.1007/s00726-016-2359-0
Weinger JG, Greenberg ML, Matheu MP et al (2015) Two-photon imaging of cellular dynamics in the mouse spinal cord. J Vis Exp. https://doi.org/10.3791/52580
Girgis NM, Gundra UM, Ward LN et al (2014) Ly6C(high) monocytes become alternatively activated macrophages in schistosome granulomas with help from CD4+ cells. PLoS Pathog 10:e1004080. https://doi.org/10.1371/journal.ppat.1004080
Matheu MP, Cahalan MD, Parker I (2011) Immunoimaging: studying immune system dynamics using two-photon microscopy. Cold Spring Harb Protoc 2011:pdb.top99. https://doi.org/10.1101/pdb.top99
Acknowledgments
We thank Simon Bartlett for English editing. Work in the laboratory of VA is supported by grants from the Spanish Ministerio de Ciencia e Innovación (MCIN)/Agencia Estatal de Investigación (AEI)/10.13039/501100011033 (PID2019-108489RB-I00) with co-funding from Fondo Europeo de Desarrollo Regional (“A way to build Europe”), Fundació la Marató TV3 (grant 202033.31), the Progeria Research Foundation (award PRF 2019-77), and Asociacion Progeria Alexandra Peraut. Work in the laboratory of JMGG is supported by grants from ISCIII (PI20/00306) and imas12 (INVESTIGA12), with co-funding from Fondo Europeo de Desarrollo Regional (“A way to build Europe”). The CNIC is supported by the MICIN, the ISCIII, and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033).
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1 Electronic Supplementary Material(s)
Supplementary video 1
Representative video of a monocyte (Ly6C, cyan) adhered to the wall of a venule in the mouse cremaster muscle. Neutrophils (Ly6G, red) were stained to identify bona fide monocytes properly. Mice are stimulated with TNF-α (0.5 mg in 0.3 mL isotonic saline) for 4 h (ZIP 21304 kb)
Supplementary video 2
Representative video of monocytes (Ly6C, cyan) rolling over the wall of a venule in the mouse cremaster muscle. Neutrophils (Ly6G, red) were stained to identify bona fide monocytes properly. Mice are stimulated with TNF-α (0.5 mg in 0.3 mL isotonic saline) for 4 h (ZIP 21619 kb)
Supplementary video 3
Representative video of a monocyte (cyan) crawling on the wall of a venule in the mouse cremaster muscle. Neutrophils (red) were stained to identify bona fide monocytes properly. Mice are stimulated with TNF-α (0.5 mg in 0.3 mL isotonic saline) for 4 h (ZIP 12118 kb)
Supplementary video 4
Representative video of migrated monocytes (green cells outside the vessel wall). Monocytes were detected using a CX3CR1-GFP reporter system. Mice are stimulated with TNF-α (0.5 mg in 0.3 mL isotonic saline) for 4 h (ZIP 7331 kb)
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Gonzalez-Granado, J.M., Del Monte-Monge, A., Piqueras, L., Andres, V., Rius, C. (2023). Analysis of Monocyte Recruitment During Inflammation by Intravital Imaging. In: Margadant, C. (eds) Cell Migration in Three Dimensions. Methods in Molecular Biology, vol 2608. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2887-4_25
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DOI: https://doi.org/10.1007/978-1-0716-2887-4_25
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