Tracking the Spatial and Functional Gradient of Monocyte-To-Macrophage Differentiation in Inflamed Lung

Abstract

Myeloid-derived cells such as monocytes, dendritic cells (DCs), and macrophages are at the heart of the immune effector function in an inflammatory response. But because of the lack of an efficient imaging system to trace these cells live during their migration and maturation in their native environment at sub-cellular resolution, our knowledge is limited to data available from specific time-points analyzed by flow cytometry, histology, genomics and other immunological methods. Here, we have developed a ratiometric imaging method for measuring monocyte maturation in inflamed mouse lungs in situ using real-time using 2-photon imaging and complementary methods. We visualized that while undifferentiated monocytes were predominantly found only in the vasculature, a semi-differentiated monocyte/macrophage population could enter the tissue and resembled more mature and differentiated populations by morphology and surface phenotype. As these cells entered and differentiated, they were already selectively localized near inflamed airways and their entry was associated with changes in motility and morphology. We were able to visualize these during the act of differentiation, a process that can be demonstrated in this way to be faster on a per-cell basis under inflammatory conditions. Finally, our in situ analyses demonstrated increases, in the differentiating cells, for both antigen uptake and the ability to mediate interactions with T cells. This work, while largely confirming proposed models for in situ differentiation, provides important in situ data on the coordinated site-specific recruitment and differentiation of these cells and helps elaborate the predominance of immune pathology at the airways. Our novel imaging technology to trace immunogenic cell maturation in situ will complement existing information available on in situ differentiation deduced from other immunological methods, and assist better understanding of the spatio-temporal cellular behavior during an inflammatory response.

Bronchus-associated macrophages efficiently capture and present soluble inhaled antigens and are capable of local Th2 cell activation

“…By optimizing methodology to minimize the spectral overlap of GFP and YFP (see Methods section for details), we confirmed that bright CX3CR1-GFP + and bright CD11c-YFP + cells were largely non- overlapping populations. When we increased the image contrast, we observed that some of the bright CX3CR1-GFP+ cells did have dim expression of CD11c-YFP, and vice versa, as expected from previous studies (Jung et al, 2000; Sen et al, 2016) (Suppl. Fig.…”

“…These cells were proposed to represent differentiated monocyte- derived cells, and we speculate that the rare cells we observed that were bright for CX3CR1- GFP and CD11c-YFP reporters in the naïve state may also represent cells that had recently differentiated from monocytes. One challenge, however, in relating this study to our findings presented here is that the high expression of CX3CR1-GFP in resident BAMs versus the lower expression in monocytes was not considered in the analysis of the ratio of CX3CR1-GFP to CD11c-mCherry expression, which formed the basis of the monocyte differentiation model (Sen et al, 2016). Similar to BAMs and DCs, monocyte-derived cells have been reported to activate T cells and drive allergic inflammation (Plantinga et al, 2013).…”

“…Although the MAR-1 antibody to FcεRI was thought to be a marker of these monocyte-derived cells, we recently established that this antibody also binds to CD64 and FcγRIV (Tang et al, 2019), and thus would also detect resident CD64 + macrophages. One study used an extensive analysis of the ratio of expression of CX3CR1-GFP and CD11c-mCherry reporters to determine the relative differentiation state of infiltrating monocytes (Sen et al, 2016). This paper showed an increase in the abundance of cells with high expression of both CX3CR1-GFP and CD11c-mCherry reporters in allergic airway inflammation models.…”

Lipopolysaccharide inhalation recruits monocytes and dendritic cell subsets to the alveolar airspace

“…This fits with numerous observations in mouse that monocyte-derived cells accumulate in tissues under inflammatory conditions and can adopt DC characteristics 3,5,20,23,25 . In vivo data suggests it takes at least 24–48 h for monocytes to differentiate into DCs 39 and in vitro, generation of DCs from monocytes (human) or bone-marrow derived cells (mouse) takes 5–7 days 40,41 . In view of our 8-h time-course, the rapid accumulation of CD1c + DCs into alveolar airspace following LPS inhalation is most in keeping with recruitment from blood and is consistent with previous observations of CD1c + DCs accumulating in bronchial (conducting airway) mucosa within 4 h of allergen challenge 35 , though detailed analyses of the infiltrating population was not possible in this earlier study.…”

Use of Integrated Optical Clearing and 2-Photon Imaging to Investigate Sex Differences in Neuroimmune Interactions After Peripheral Nerve Injury

“…Lastly, we assessed sphericity of LysM tdT+ macrophages after nerve injury ( Figure 3B ). Although we find no significant differences, cells that take on a more prolate (flattened) shape after injury are also more spherical, indicating a more active phenotype (Sen et al, 2016 ). Taken together, we conclude that macrophages have a dynamic morphological response to injury and may be characterized based on their geometric shape.…”