A Polecat’s View of Patrolling Monocytes

Abstract

A major obstacle to intravital visualization of inflammatory processes within large arteries is the motion of vessels that occurs near the beating heart. In this issue, Quintar et al. apply ILTIS, the German word for the European polecat that serves as an acronym for Intravital Live-cell Triggered Imaging System, to capture the most sophisticated images of inflammatory cell dynamics in the arterial vasculature to date, by timing data acquisition to a consistent point in every heartbeat. The authors show that patrolling nonclassical monocytes scan endothelium in plaque-prone areas, much as they have been described to elsewhere. The inability of these monocytes to patrol arteries leads to heightened endothelial damage within the artery. In this way, nonclassical monocytes safeguard against plaque development and progression.

As precursors for foam cells, monocytes are key mediators of atherosclerotic disease, making them suitable targets for clinical therapeutic intervention¹. In mice, monocytes come in two principal forms; ‘classical’ defined as Ly6C^hi CCR2^hi CD43^low that are CX3CR1-GFP^dim or ‘nonclassical’ defined Ly6C^low CCR2^low CD43^hi that are CX3CR1-GFP^bright, with both sharing common monocyte lineage markers CD45, CD11b, and CD115^{2, 3}. Analogous CD14⁺ CD16⁻ classical and CD14^dim CD16⁺ nonclassical monocyte subset have also been described in humans, and were shown to have similar gene expression profiles in the two species⁴, although the nonclassical subset is rare in humans relative to mice. Classical monocytes are rather short-lived cells with an approximate half-life of 20 hours before they either enter tissue or mature into the nonclassical monocytes. Classical monocytes are the major cell infiltrating into the intimal wall^{5, 6}, which are known to contribute to the formation of lipid-laden foamy macrophages. Research has primarily focused on the role of the classical monocyte, as they enter tissue in the steady state and during inflammation. Classical monocytes mature to nonclassical monocytes within the vasculature, where they have a lifespan of 3–8 days, depending on the status of the overall monocyte pool⁷.

While the main function of nonclassical monocytes has been elusive, past ideas from the laboratory of Frederic Geissmann suggested that they might serve as the major precursor for tissue resident macrophages⁸ or were precursors for alternatively activated macrophages during inflammation. These concepts have been discarded in favor of clearer evidence that they scarcely leave the vasculature at all, but have tendency to patrol vessels, at least in the various microvascular beds examined to date⁹. Subsequently, Carlin et al. proposed that their tendency to scan the vasculature was prone to support endothelial damage because, while patrolling endothelium, nonclassical monocytes recruited neutrophils to kill endothelial cells¹⁰. However, it is difficult to conceive that a white blood cell type evolved to support endothelial damage. Instead, the field quickly picked up on the possibility that the scanning of the vasculature by nonclassical monocytes might allow them, with some exceptions such as when they attracted and activated neutrophils, to promote endothelial integrity. With this evolved viewpoint, it made sense that deficiency of NR4A1, the transcription factor that nonclassical monocytes depend upon for maturation, led to worsened atherosclerotic plaques^{11, 12}. The interpretation was that the absence of endothelial protection by nonclassical monocytes rendered endothelium even more susceptible to plaque growth. However, what was missing to support this interpretation was direct evidence that nonclassical monocytes actually patrol the areas of the vasculature where plaques form. Indeed, it was completely unclear if flow dynamics in larger arteries were permissible to patrolling by nonclassical monocytes. Clarity now arrives with the present work of Quintar et al., who took on this conceptual problem, even though doing so required overcoming substantial technical challenges.

The high shear stress and pulsatile flow environment of the mid- and large-sized arteries creates a technically difficult location to obtaining high resolution live imaging at the carotid artery. The Ley lab previously introduced the Intravital Live cell Triggered Imaging System (ILTIS)¹³ (Fig. 1), which, in an original approach, avoids the vibration noise from the pumping heart. The ILTIS system senses the pulse in the periphery using a cuff on the animal’s thigh, allowing longitudinal imaging that is synchronized with cardiac rhythm. This increases image stability and reduces movements that would otherwise affect images collected using conventional intravital approaches, where images are collected in fixed time intervals unlinked to cardiac rhythm. Importantly, using the ILTIS system creates a scenario where direct imaging can be performed without compression being placed on the vessel wall as a strategy to limit unwanted motion, allowing for the most-unperturbed approach currently available. Further, a nice tool developed by the authors utilizes a Matlab script to smooth artifacts created by residual shaking that remains after application of ILTIS, further enhancing the video and image quality.

Figure 1.

Using ILTIS, depicted by a polecat on the right (iltis is German for polecat), nonclassical monocytes, studied in CX3CR1-GFP reporter mice, can be visualized in the carotid artery performing patrolling functions along the endothelium. Numbers of patrolling monocytes was dramatically increased in animals fed a western diet compared to chow-fed controls.

In their approach, emission light was separated by the use of traditional bandpass filter sets (460/40, 513/50, and 640/40), which we speculate might explain why the classical monocyte population CX3CR1-GFP^dim could not be visualized, even when FACS sorted and imaged ex vivo on dishes. For the current study, this selective visualization of only one monocyte subset is in fact advantageous as the authors could identify the nonclassical population without confusion over which cells were being tracked. In our experience, classical monocytes can be visualized by 2-photon microscopy approaches when emission light is directed immediately over the objective to external chilled hybrid detectors and then separated by high efficiency single-edge dichroic beam splitters without the use of any bandpass filters. Using such a combination of mirrors in the future should allow for the identification of the classical monocyte populations to address the possibility that they also patrol the arterial vasculature. Furthermore, imaging classical monocytes in combination with the ILTIS approach would allow assessment of the anticipated recruitment of classical monocytes in atherosclerotic lesions.

As an additional technical note, it might be surprising to the reader that much of the study was done in arteries that do not bear atherosclerotic plaque. We speculate, based on our own experience with similar preparations for imaging, that this may be due in part to the location of where plaque typically form at the carotid bifurcation that is hard to reach in preparations. It also remains unclear how possible it is to image within highly developed plaques by necessarily approaching plaques from the adventitial side of the vessel.

With the unique ILTIS setup, the authors address three distinct scenarios to help understand the role of nonclassical monocytes in patrolling large arteries: (i) WT, atherosclerosis-resistant mice fed a normal chow diet, (ii) Western diet-fed WT animals that are not susceptible to atherosclerotic lesions but would be expected to develop features of metabolic syndrome, and (iii) ApoE^−/− mice fed a Western diet to promote hypercholesterolemia and atherosclerotic lesion development. Under any of these conditions, Quintar et. al. observed that monocyte movement occurred at 3 times the speed earlier reported in the peripheral microvasculature⁹ or as measured in the ear microcirculation. Using ILTIS, the temporal resolution of movies used to calculate nonclassical monocyte motility is approximately 1 second between frames. This is superb compared to conventional 2-photon approaches that often collect at longer intervals. However, this increased frame rate must be taken into account when comparing velocities calculated from imaging performed at slower rates. The increased rate will capture additional movements that will be missed by slower frame rates¹⁴.

In response to a short exposure to western diet, WT mice showed dramatic increases in the number of interacting nonclassical monocytes at the carotid endothelium, but no major shifts in rolling behavior (Fig. 1). The heightened nonclassical monocyte patrolling may be due to endothelial damage induced by metabolic challenge in the WT animals, though this issue was not explicitly addressed. The observed increase in endothelial – monocyte interactions was even more robust in atherosclerotic conditions in the ApoE^−/− mice.

To address functional characteristics of the nonclassical monocyte, the authors employed the use of known blocking antibodies to LFA-1 and VLA-4. Following TLR7-mediated activation to promote nonclassical monocyte recruitment to the endothelial layer, treatment with these inhibitors showed only partial loss of monocyte adherence. Interestingly, the monocytes that did successfully bind the endothelium showed normal migratory kinetics compared to controls. This along with their observation that CX3CR1 was dispensable for nonclassical monocyte adhesion and patrolling is consistent with but differing from published work showing an absolute necessity for LFA1 and at least partial dependency on CX3CR1 pathway for crawling behavior in the mesenteric vessels⁹. These data suggest that unique or perhaps more complex mechanisms are at play in the large artery endothelium to regulate the patrolling behavior of nonclassical monocytes.

To investigate the potential ‘housekeeper’ function of nonclassical monocytes for arterial endothelium, Quintar et al. turned to the examination of the endothelium by electron microscopy. In the absence of the nonclassical subset (Nr4a1^−/−), Quintar et. al. show that HFD-mediated endothelial damage is elevated in ApoE^−/− mice. This observation brings the earlier work from the Hedrick laboratory^{11, 12} full circle, suggesting strongly, and now directly, that indeed nonclassical monocytes protect, rather than harm, the endothelium, likely accounting for their reported atheroprotective role. The use of electron microscopy to assess the status of the endothelium is consistent with the assessment strategy of Carlin et al. However, the assessment is rather nonspecific and the exact nature of the endothelial cell damage remains unclear. Likewise, it is extremely vague as to what contact with nonclassical monocytes does to preserve endothelial integrity. These problems must now be addressed, especially given that the human counterparts of mouse nonclassical monocytes are rather rare and may be insufficient to provide the vascular protection needed under high risk circumstances. Knowing precisely what signals, if any, pass between monocyte and endothelium in the process of protection will be crucial to develop translational applications. Can the ILTIS begin to provide answers to these new directions? We certainly look forward to finding out. Like the nocturnal European polecat, the iltis, we wait eagerly in the dark for answers to come to light.