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PLoS One
2016 Oct 03;1110:e0165064. doi: 10.1371/journal.pone.0165064.
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Tracking the Spatial and Functional Gradient of Monocyte-To-Macrophage Differentiation in Inflamed Lung.
Sen D
,
Jones SM
,
Oswald EM
,
Pinkard H
,
Corbin K
,
Krummel MF
.
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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.
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Fig 1. A dual reporter system reveals maturation of monocytes inside and outside of the lung vasculature.(A) Illustration of the Cx3cr1-eGFP-Cd11c-mCherry transgenic mouse model used for lineage tracing of monocyte to DCs. (B) Flow cytometry with absolute numbers (insets) and (C) normalized total counts of GFP+ and/or Cherry+ cells in saline, Ova or HDM-treated lungs within a CD45+CD11b+Siglec-F-Ly6G- gate. Data in C was normalized to input fluorescent polystyrene beads during lung digests. Data in B-C represent 3 independent experiments. (D) Experimental layout and results of intravenous labeling of blood monocytes as a pulse with intravenous beads (“pulse”), followed by tracking of bead+ lung-homing monocytes or monocyte-derived cells over 48 hours during the “chase” period. Flow cytometry data is plotted as a ratio of the GFP and mCherry channel with gates for ‘low’ set based on Saline at -24 hours, ‘med’ based on OVA at -24 hours and ‘hi’ representing populations that appear predominantly after 48 hours. Data represent 3 independent experiments. (E) Experimental layout and resultant flow cytometric analysis of intravenous labeling of cells with high molecular weight anti-CD45 antibodies 5 minutes prior to euthanasia. CD45+CD11b+Siglec-F-Ly6G- lung monocytic cells are plotted on the identical Cherry:GFP scale as (D), with the CD45 i.v. specifically labeling intravenous immune populations as delineated in S2B Fig. (F) Illustration of the extent of monocyte→myeloid maturation and concomitttant tissue distribution that is differentiated by ratiometric dual reporting of Cx3cr1-eGFPxCd11c-mCherry mice. Data in D-F representative of at least three mice per condition.
Fig 2. Airway-proximal accumulation of myeloid populations in inflamed mouse lungs.Mouse lungs were sectioned and analyzed using the color labeling strategy defined in Fig 1. Bins were set similarly to flow, using the transformation shown in S3 Fig. (A,D,G) Two-photon micrographs of control (A) Ova- (D) or HDM (G) treated lungs from Cx3cr1-eGFP x Cd11c-mCherry mice. Scale bar = 100 μm. ‘AW’ = airway. (B,E,H) Distances of myeloid cells from nearest airway epithelium in control (B) ova- (E) or HDM (H) treated lungs. Myeloid populations were categorized into divisions based on the imaging Cherry:GFP color scale, as described in S3 Fig. (C,F,I) Density of myeloid populations in control (C) ova- (F) or HDM (I) treated lungs, as a function of distances from airways, using the identical ratio color scale as in panels B,E,H. See also Video 1. Data represent 4 independent experiments for each condition.
Fig 3. Increased sphericity and reduced motility of monocyte-derived populations as they differentiate in inflamed mouse lungs.(A,C) Sphericity of GFP+ leukocytes in control (A) or Ova-treated (B) lungs categorized into divisions based on Cherry:GFP ratio score or Cherry+only cells and AMs. (B,D) Normalized trajectories of cells in panels A and B respectively (Also see S1 Video). (E,F) Track speed mean of cells in panels A and B respectively. * p<0.05; ** p<0.005; *** p<0.001. (Pairwise t-test compared to ratio low cells). (G) “Flower” displacement plots of 20 randomly selected tracks from each differentiation bin. (H) Track displacements of cells from panel A with respect to the airway. Positive numbers indicate movement towards, and negative numbers away from the airway. * p<0.05. All others are pairwise not significant at the 95% confidence interval (Unpaired t-test). Data in A-H are from 4 independent experiments.
Fig 4. In situ maturation of CX3CR1+ monocyte-derived cells within inflamed lungs.(A) Two-photon micrographs at three selected time points demonstrating in situ upregulation of the mCherry signal and thus advancement in red/green ratio. Top row shows an extended myeloid cell, showing Cherry (red) and GFP (green) channels. Bottom row shows rendered isosurfaces of the same cells, using a Cherry:GFP ratio-based color scale. Note: 0h is the beginning of imaging on day 24 of ova treatment. Scale bar = 20 μm. Also see Video 2. (B) Cherry:GFP ratio, normalized to the first frame, for 3 exemplar moDCs along with similar plots for 2 alveolar macrophages (AM) for comparison. Linear fits are shown by dotted lines, Each color represents an individual cell. (C) Similar plots for three cells taken from control lungs, also compared to exemplar AMs and showing reduced in situ maturation. (D) Plots of change in Cherry:GFP ratio normalized to AM intensity and time, for multiple cells studied in OVA-challenged and control lungs showing statistically significant p<0.05 differences in allergic lungs as compared to control. Data represent 3 independent experiments.
Fig 5. Antigen uptake and T cell engagement increase for differentiated monocytes.(A) Protocol for inhalation of fluorescent VPD450+ HDM to score for efficiency of antigen capture in myeloid cells in situ. Labeled HDM was administered i.n. on day 16 in a protocol which otherwise closely resembled that outlined in S1 Fig. (B) Flow histogram showing distribution of HDM taken up by CD45+CD11b+Siglec-F-Ly6G- lung leukocytes on the Cherry:GFP color scale, with percentages of VPD450+ cells shown above and demonstrating dominance of uptake by the most differentiated cells. (C) Two photon micrograph of HDM-treated lungs showing GFP+ and/or Cherry+ cells. Note blue positivity for multiple populations and across the lung. Scale bar = 100 μm. Right: XY and XZ sectional views of the regions marked in C showing HDM particles within moDCs (white arrow, bound by blue) and accumulation of ratio-mid and ratio-hi cells around HDM particles (beads appear purple, region of interest indicated by dotted circles). Scale bars = 20 μm. (D, E) Quantification of endocytosed HDM observed by two-photon microscopy, shown as (D, histogram; excluding Cherry+only cells) or including (E, pie chart; including Cherry+only cells). (F) Contact duration between adoptively transferred and labeled OTII T cells and the indicated myeloid population at 16 hour post T cell transfer in a similar OVA-model for antigen uptake (see S4 Fig), derived from timelapse imaging of lungs as previously described[11]. Dwell times > 4 min are considered. All data taken from at least 3 experiments.
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