Label-free imaging of M1 and M2 macrophage phenotypes in the human dermis in vivo using two-photon excited FLIM
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Curated by eLife
Evaluation Summary:
Kröger et al use 2-photon FLIM tomography to perform correlative imaging on in vitro, ex vivo and in vivo blood and skin cells to determine characteristic NADPH fluorescence lifetimes for M1 and M2 ends of macrophage spectrum. Interestingly, M1 and M2 macrophages, and all other tissue cells, had distinctive lifetime features leading to robust prediction of phenotypes, with ground trust defined by cytokine staining. They generate a decision tree that has ~90% accuracy in identifying M1 and M2 based on FLIM parameters and additional information. The ability to use two photon fluorescence lifetime tomography of NADPH fluorescence to identify macrophages and their inflammatory status in human tissues should open opportunities in experimental medicine and eventually medical diagnosis.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Macrophages (ΜΦs) are important immune effector cells that promote (M1 ΜΦs) or inhibit (M2 ΜΦs) inflammation and are involved in numerous physiological and pathogenic immune responses. Their precise role and relevance, however, are not fully understood for lack of noninvasive quantification methods. Here, we show that two-photon excited fluorescence lifetime imaging (TPE-FLIM), a label-free noninvasive method, can visualize ΜΦs in the human dermis in vivo. We demonstrate in vitro that human dermal ΜΦs exhibit specific TPE-FLIM properties that distinguish them from the main components of the extracellular matrix and other dermal cells. We visualized ΜΦs, their phenotypes and phagocytosis in the skin of healthy individuals in vivo using TPE-FLIM. Additionally, machine learning identified M1 and M2 MФs with a sensitivity of 0.88±0.04 and 0.82±0.03 and a specificity of 0.89±0.03 and 0.90±0.03, respectively. In clinical research, TPE-FLIM can advance the understanding of the role of MФs in health and disease.
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Evaluation Summary:
Kröger et al use 2-photon FLIM tomography to perform correlative imaging on in vitro, ex vivo and in vivo blood and skin cells to determine characteristic NADPH fluorescence lifetimes for M1 and M2 ends of macrophage spectrum. Interestingly, M1 and M2 macrophages, and all other tissue cells, had distinctive lifetime features leading to robust prediction of phenotypes, with ground trust defined by cytokine staining. They generate a decision tree that has ~90% accuracy in identifying M1 and M2 based on FLIM parameters and additional information. The ability to use two photon fluorescence lifetime tomography of NADPH fluorescence to identify macrophages and their inflammatory status in human tissues should open opportunities in experimental medicine and eventually medical diagnosis.
(This preprint has been reviewed by …
Evaluation Summary:
Kröger et al use 2-photon FLIM tomography to perform correlative imaging on in vitro, ex vivo and in vivo blood and skin cells to determine characteristic NADPH fluorescence lifetimes for M1 and M2 ends of macrophage spectrum. Interestingly, M1 and M2 macrophages, and all other tissue cells, had distinctive lifetime features leading to robust prediction of phenotypes, with ground trust defined by cytokine staining. They generate a decision tree that has ~90% accuracy in identifying M1 and M2 based on FLIM parameters and additional information. The ability to use two photon fluorescence lifetime tomography of NADPH fluorescence to identify macrophages and their inflammatory status in human tissues should open opportunities in experimental medicine and eventually medical diagnosis.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Public Review:
In "Label-free imaging of macrophage phenotypes and phagocytic activity in the human dermis in vivo using two-photon excited FLIM", Kroger et al attempt to visualize macrophages and distinguish their phenotypes using FLIM within the skin of live animals. This study provides data characterizing the fluorescence lifetime signatures of macrophages derived from peripheral blood mononuclear cells or dermal macrophages stimulated with IFNg or IL4, to polarize them towards more M1-like and M2-like phenotypes, respectively. The FLIM signatures are compared to macrophages in other conditions or other cell types, including macrophages ex vivo in skin cryosections, macrophages in forearm of healthy human individuals, as well as mast cells, dendritic cells, fibroblasts, and neutrophils in vitro. The authors then use …
Public Review:
In "Label-free imaging of macrophage phenotypes and phagocytic activity in the human dermis in vivo using two-photon excited FLIM", Kroger et al attempt to visualize macrophages and distinguish their phenotypes using FLIM within the skin of live animals. This study provides data characterizing the fluorescence lifetime signatures of macrophages derived from peripheral blood mononuclear cells or dermal macrophages stimulated with IFNg or IL4, to polarize them towards more M1-like and M2-like phenotypes, respectively. The FLIM signatures are compared to macrophages in other conditions or other cell types, including macrophages ex vivo in skin cryosections, macrophages in forearm of healthy human individuals, as well as mast cells, dendritic cells, fibroblasts, and neutrophils in vitro. The authors then use immunohistochemistry to correlate the FLIM signatures with phenotype markers, CD68 and CD163. Finally, the authors visualize phagocytosis through morphological changes and identify FLIM signatures of phagocytic macrophages.
Strengths:
Using optical methods to non-invasively detect cells has significant interest for clinical and basic studies, and the impact of this study is considered high.
The authors have identified different FLIM signatures for macrophages polarized towards different phenotypes in vitro, and were able to compare these signatures to those of other cell types and macrophages in the skin.
They identified a few cells in the skin that expressed markers associated with macrophage polarization, and also exhibited the FLIM signatures that were established from the in vitro polarization studies.
Weaknesses:
There are some significant technical concerns given that macrophages are a highly heterogeneous population of cells, particularly in vivo during an activation event such as injury. The few cells analyzed in Figure 3 are not sufficient given the heterogeneity of macrophages in vivo. Mixed phenotypes are common in vivo, and it is unclear how the FLIM signatures would correlate to such mixed signatures.
Visualizing a single phagocytosing cell in Figure 5 is also not sufficient to conclude that the method is capable of detecting phagocytosis events.
Finally, the reporting of lifetime alone does not offer insights into the function of the macrophages. The study would be strengthened with further analysis that correlates FLIM signatures with metabolic state (free vs. bound NADPH).
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