Differential roles of microglia and monocytes in the inflamed central nervous system

Microglia derive from yolk-sac progenitors during embryogenesis.

“Microglia and monocytes are ontogenetically distinct: microglia derive from yolk-sac progenitors during embryogenesis (Ginhoux et al., 2010; Schulz et al., 2012)”

• cites Ginhoux et al., 2010 sourceSupported
• cites Schulz et al., 2012 sourceNot checked — no full text of the source

In MS autopsy tissues, prominent macrophage accumulation correlates with active demyelination.

“In MS autopsy tissues, prominent macrophage accumulation correlates with active demyelination (Ferguson et al., 1997; Trapp et al., 1998).”

• cites Ferguson et al., 1997 sourceNot checked — no full text of the source
• cites Trapp et al., 1998 sourceSupported

EAE models inflammatory aspects of multiple sclerosis.

“In EAE, which models inflammatory aspects of MS (Williams et al., 1994; Ransohoff, 2012), macrophages dominate the inflammatory infiltrates and their numbers correlate to EAE severity (Huitinga et al., 1990, 1993; Ajami et al., 2011).”

• cites Williams et al., 1994Not checked — no full text of the source
• cites Ransohoff, 2012 sourceSupported

It is estimated that 30-50% of activated macrophages in active MS lesions derive from microglia.

“Based on kinetics of cell accumulation and differential marker expression, it’s estimated that 30–50% of activated macrophages in active MS lesions derive from microglia (Brück et al., 1995; Trebst et al., 2001).”

• cites Brück et al., 1995Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Trebst et al., 2001 sourcePartly supported

  The section on hematogenous monocytes and resident microglia during early demyelination (page unavailable) supports a microglial contribution, but estimates that 58–75% of CD68-positive phagocytes in early-active zones most plausibly represent resident microglia—not 30–50%. The paper does not give the claimed percentage for activated macrophages across active MS lesions. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

CCR2 is essential for monocyte recruitment to CNS tissues during immune-mediated inflammation.

“CCR2 is essential for monocyte recruitment to CNS tissues during immune-mediated inflammation (Fife et al., 2000; Izikson et al., 2000; Savarin et al., 2010).”

• cites Fife et al., 2000 sourceSupported
• cites Izikson et al., 2000 sourcePartly supported

  The Results and Discussion report that CCR2-deficient mice did not develop the CNS infiltrates seen in wild-type mice with experimental autoimmune encephalomyelitis, and suggest that CCR2 signaling is needed to recruit monocytes into the brain and spinal cord. The claim omits that this evidence comes from a mouse model and states the proposed recruitment mechanism as a general requirement during immune-mediated CNS inflammation. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites Savarin et al., 2010 sourcePartly supported

  The Discussion (page unavailable) reports a drastic reduction in CNS monocytes in CCR2-deficient mice during JHMV infection. That supports a role for CCR2 in monocyte recruitment during viral encephalitis, but does not establish that CCR2 is essential across immune-mediated CNS inflammation generally. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

In Ccr2rfp/rfp::Cx3cr1gfp/+ mice, MDMs were virtually absent from inflamed EAE tissues and replaced in large part by neutrophils.

“To address the role of MDMs in demyelination at EAE onset, we investigated clinical characteristics in relation to node pathology and demyelination in Ccr2rfp/rfp::Cx3cr1gfp/+ mice in which MDMs were virtually absent from inflamed EAE tissues and replaced in large part by neutrophils (Saederup et al., 2010).”

• cites Saederup et al., 2010 sourcePartly supported

  The Discussion (page unavailable) says Ly6Chi monocytes were virtually absent from the CNS of these mice and that neutrophils replaced them. The Results section on CD115+ infiltration reports markedly fewer monocytes/macrophages, but also says Ly6Clo cells remained; it does not establish that all monocyte-derived macrophages (MDMs) were virtually absent. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

The study described transcription factors and miRNAs characteristic of microglia in healthy brain but not in peripheral monocytes.

“The study described transcription factors and miRNAs characteristic of microglia in healthy brain but not in peripheral monocytes.”

• cites Butovsky et al., 2014 sourceSupported

The detailed profiling after separating cells via CD45dim status showed distinct mRNA, miRNA, and protein expression by microglia as compared with infiltrating monocytes or neuroepithelial brain cells.

“The detailed profiling after separating cells via CD45dim status showed distinct mRNA, miRNA, and protein expression by microglia as compared with infiltrating monocytes or neuroepithelial brain cells (Butovsky et al., 2014).”

• cites Butovsky et al., 2014 sourcePartly supported

  The Results (‘Identification of a unique microglial signature’; page not provided) report distinct gene and protein expression in CD11b+CD45Low microglia versus splenic Ly6C+ monocytes. The paper also compares microglial genes and miRNAs with astrocytes, oligodendrocytes and neurons, but does not report a protein-expression comparison with those CNS cells. The monocytes in the principal profiling comparison were isolated from spleen, not identified as infiltrating cells in the brain. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

SBF-SEM would exhibit advantages for spatial resolution required for characterizing relationships of myeloid cells to axoglial units during the inflammatory demyelinating process at EAE onset.

“SBF-SEM would exhibit advantages for spatial resolution (Denk and Horstmann, 2004) required for characterizing relationships of myeloid cells to axoglial units during the inflammatory demyelinating process at EAE onset.”

• cites Denk and Horstmann, 2004 sourceSupported

Brisk microglial proliferation may have accelerated a gradual return toward a homeostatic expression profile, as suggested by MiDM up-regulation of fos, jun, myc, and CSF1 at disease onset.

“Brisk microglial proliferation (Ajami et al., 2011) may have accelerated a gradual return toward a homeostatic expression profile, as suggested by MiDM up-regulation of fos, jun, myc, and CSF1 (Wei et al., 2010) at disease onset.”

• cites Ajami et al., 2011 sourceNot checked — no full text of the source
• cites Wei et al., 2010 sourcePartly supported

  The Introduction (page not provided) identifies CSF-1 as a regulator of mononuclear-phagocyte proliferation and microglial development, supporting the relevance of CSF1 to the proposed proliferation. It does not report MiDM up-regulation of fos, jun, myc, or CSF1 at disease onset, or a return to a homeostatic expression profile. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

MiDM gene expression during the course of EAE did not correspond to the M1/M2 pattern of peripheral macrophage responses to infection or tissue injury.

“MiDM gene expression during the course of EAE did not correspond to the M1/M2 pattern of peripheral macrophage responses to infection or tissue injury.”

• cites Chiu et al., 2013 sourcePartly supported

  The Results section ('SOD1G93A microglia differ significantly from LPS induced microglia and M1 or M2 macrophages'; page unavailable) finds that microglia in an ALS mouse model did not show an M1 or M2 bias during disease progression. This supports the general M1/M2 comparison, but the paper examines resident microglia in ALS, not MiDM gene expression during EAE. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Microglia are uniformly CX3CR1+ from early embryonic time points through adulthood.

“microglia are uniformly CX3CR1+ from early embryonic time points through adulthood (Cardona et al., 2006; Ginhoux et al., 2010; Schulz et al., 2012)”

• cites Cardona et al., 2006 sourceNot checked — no full text of the source
• cites Ginhoux et al., 2010 sourcePartly supported

  The passage on developmental markers (section labeled Abstract; page unavailable) identifies CX3CR1 as a microglial marker and reports CX3CR1-expressing myeloid cells in the developing brain from E9.5. It does not establish that all microglia express CX3CR1 uniformly at every stage through adulthood. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites Schulz et al., 2012 sourceNot checked — no full text of the source

The authors characterized resident microglia by profiling mRNA, miRNA, and protein in comparison with infiltrated brain macrophages, nonmicroglial resident brain cells, and peripheral macrophages.

“we characterized resident microglia by profiling mRNA, miRNA, and protein in comparison with infiltrated brain macrophages, nonmicroglial resident brain cells, and peripheral macrophages (Butovsky et al., 2014).”

• cites Butovsky et al., 2014 sourceSupported

These findings partially explain a divergent response of these two cell types to the same stimuli.

“These findings partially explain a divergent response of these two cell types to the same stimuli (Butovsky et al., 2014).”

• cites Butovsky et al., 2014 sourcePartly supported

  The Results (Identification of a unique microglial signature; page unavailable) show substantial molecular differences between microglia and monocytes, which could help explain different responses. The cited paper does not establish that the two cell types respond divergently to the same stimuli; its reported stimulation experiments concern microglia rather than a direct comparison between the cell types. (LLM, conf=0.78)

  Experimental — accuracy not yet measured

CCR2+Ly6C+ cells constitute the vast majority of infiltrating monocytes at EAE onset.

“CCR2+Ly6C+ cells constitute the vast majority of infiltrating monocytes at EAE onset (Saederup et al., 2010; Mizutani et al., 2012).”

• cites Saederup et al., 2010 sourcePartly supported

  The Results (Figure 4D) report that 88% of CD115+ monocyte-lineage cells in the CNS were Ly6Chi, and another Results passage (Figure 4E) identifies this subset as CCR2+. These measurements were made at peak EAE severity, not at EAE onset, so the cited paper does not establish the claim's timing. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites Mizutani et al., 2012 sourcePartly supported

  The Results section identifies CCR2+ inflammatory Ly6Chi monocytes and describes classical infiltrating monocytes as CCR2+. It does not establish that CCR2+Ly6C+ cells make up the vast majority of infiltrating monocytes at EAE onset. The reported 90 ± 3.5% concerns peripherally derived cells among CD45hi cells at peak EAE, not the claimed monocyte subset at onset; page numbers are unavailable in our copy. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Microglia clear debris in EAE.

“Phagocytic monocyte-derived macrophages associate with the nodes of Ranvier and initiate demyelination while microglia clear debris and display a suppressed metabolic gene signature in EAE.”

Flow cytometry demonstrated distinct expression of RFP and GFP by F4/80+/CD45high MDMs and F4/80+/CD45dim MiDMs, respectively, in Ccr2rfp/+::Cx3cr1gfp/+ spinal cords at disease onset.

“Using cells isolated from Ccr2rfp/+::Cx3cr1gfp/+ spinal cords at disease onset, flow cytometry demonstrated distinct expression of RFP and GFP by F4/80+/CD45high MDMs and F4/80+/CD45dim MiDMs, respectively (Fig. 1 B).”

At onset of EAE, two pools of CD11b+ mononuclear phagocytic cells, putative red MDMs and green MiDMs, predominated in spinal cord.

“At onset of EAE, two pools of CD11b+ mononuclear phagocytic cells (putative red MDMs and green MiDMs) predominated in spinal cord (Fig. 1 A), indicating that fluorochrome markers could be distinguished at this time point.”

MDMs and MiDMs showed equal numbers at disease onset when explosive MDM accumulation occurred.

“Enumeration of cells recovered from cell sorting using F4/80+RFP+ as MDMs gate and F4/80+GFP+ as MiDMs gate indicated that MDMs and MiDMs showed equal numbers at disease onset when explosive MDM accumulation occurred.”

Microglia display a suppressed metabolic gene signature in EAE.

“Phagocytic monocyte-derived macrophages associate with the nodes of Ranvier and initiate demyelination while microglia clear debris and display a suppressed metabolic gene signature in EAE.”

Green group genes, up-regulated at onset and transiently further up-regulated at peak, were comprised mainly of complement-system elements, mononuclear cell-specific chemokines, proliferation related genes, and acute inflammation-related genes.

“Green group genes, up-regulated at onset and transiently further up-regulated at peak, were comprised mainly of complement-system elements (C3aR1; C4a, C1qa, C1qa, C3, and Cfb); mononuclear cell–specific chemokines (CCl2, 3, 4, 5, 7, and CXCL9); proliferation related genes (fos, jun, myc, and CSF1); and acute inflammation–related genes (IL1a, IL1b, TNF, CEBP, STAT1).”

If one cell type is present (20/75 cells), it is nearly always (18/20 segments) MDMs.

“If one cell type is present (20/75 cells), it’s nearly always (18/20 segments) MDMs.”

In an equivalent sample of EAE tissues from three Ccr2rfp/rfp::Cx3cr1gfp/+ mice, only one MDM was found in contact with a node of Ranvier, despite the presence of disrupted nodes in proximity to neutrophils.

“In an equivalent sample of EAE tissues from three Ccr2rfp/rfp::Cx3cr1gfp/+ mice, only one MDM was found in contact with a node of Ranvier, despite the presence of disrupted nodes in proximity to neutrophils.”

In one instance, a neutrophil abstracting myelin from the myelin internode was detected despite a nearby disrupted node in tissues from a Ccr2rfp/rfp::Cx3cr1gfp/+ mouse.

“In one instance, we detected a neutrophil abstracting myelin from the myelin internode (Fig. 5, left and right) despite a nearby disrupted node (Fig. 5, left) in tissues from a Ccr2rfp/rfp::Cx3cr1gfp/+ mouse.”

Several 3D shape parameters discriminated between MDMs and MiDMs.

“Several 3D shape parameters also discriminated between MDMs and MiDMs (Fig. 1 D).”

There was no evidence for neutrophil recognition of disrupted nodes of Ranvier.

“Importantly, there was no evidence for neutrophil recognition of disrupted nodes of Ranvier.”

MDM-enriched genes were sharply up-regulated from naive monocytes to onset and peak-stage MDMs, descending toward naive levels during recovery.

“MDM-enriched genes were sharply up-regulated from naive monocytes to onset and peak-stage MDMs (Fig. 6 B), descending toward naive levels during recovery (Fig. 6 B).”

EAE onset tissues of Ccr2rfp/rfp::Cx3cr1gfp/+ mice were characterized by nodal pathology often without cellular infiltrates.

“By distinct contrast, EAE onset tissues of Ccr2rfp/rfp::Cx3cr1gfp/+ mice were characterized by nodal pathology often without cellular infiltrates (Fig. 5 D).”

Where only one myeloid cell type was present (20/75; 27%), nearly all axoglial units made contacts to MDMs.

“Where only one myeloid cell type was present (20/75; 27%), nearly all axoglial units made contacts to MDMs (Fig. 2).”

Remaining myelin was undergoing vesicular breakdown.

“Remaining myelin was undergoing vesicular breakdown (Fig. 3 A).”

Most (55/75; 73%) of all segments, both intact and demyelinated, contacted both MDMs and MiDMs.

“We quantified contacts made by MDMs (n = 169) and MiDMs (n = 86) with axoglial units (n = 75; Fig. 2), and observed that most (55/75; 73%) of all segments (both intact and demyelinated) contacted both MDMs and MiDMs (Fig. 2).”

An MDM apposed to a node of Ranvier and actively phagocytizing myelin was identified.

“Additionally we identified an MDM apposed to a node of Ranvier and actively phagocytizing myelin (Fig. 4 C).”

MDMs have close relationship with nodes of Ranvier (7 MDMs /75 axoglial units).

“MDMs have close relationship with nodes of Ranvier (7 MDMs /75 axoglial units).”

Of the MDMs found in sole contact with axoglial units, virtually all (>90%) MDMs contained myelin when found in sole contact with a demyelinated axon.

“Of the MDMs found in sole contact with axoglial units, virtually all (>90%) MDMs contained myelin when found in sole contact with a demyelinated axon (Fig. 2).”

In SBF-SEM data, only MDMs seemed to be implicated in active damage to myelin.

“In our SBF-SEM data, only MDMs seemed to be implicated in active damage to myelin.”

The myelin peeled away from the axon remained in continuity with a large myelin inclusion inside the MDMs.

“The myelin peeled away from the axon remained in continuity with a large myelin inclusion inside the MDMs (Fig. 3 A).”

Reconstruction of one representative image stack shows MDMs with large intracellular myelin inclusions tightly encircling a partially demyelinated axon.

“Reconstruction of one representative image stack shows MDMs with large intracellular myelin inclusions tightly encircling a partially demyelinated axon (Fig. 3 A).”

None of the five gene groups showed coordinate regulation patterns in MDMs, as were observed in MiDMs.

“None of the gene groups showed coordinate regulation patterns in MDMs, as were observed in MiDMs (Fig. 6 H).”

k-means clustering discriminated five distinct patterns of MiDM gene expression during the course of EAE.

“We used k-means clustering to discriminate five distinct patterns of MiDM gene expression during the course of EAE (Fig. 6, E and F).”

Only 18/48 (37.5%) of MiDM-enriched genes encoded effector functions.

“In contrast, only 18/48 (37.5%) of MiDM-enriched genes encoded effector functions (Fig. 6 D, Table S1, purple genes).”

MiDM expansion began at peak.

“MiDM expansion began at peak (Fig. 1 B).”

In three distinct tissues from individual Ccr2rfp/+::Cx3cr1gfp/+ mice with EAE preonset, five MDMs attached to disrupted nodes of Ranvier were found.

“In three distinct tissues from individual Ccr2rfp/+::Cx3cr1gfp/+ mice with EAE preonset, we found five MDMs attached to disrupted nodes of Ranvier.”

2-3 MDMs attached to each of the 18/20 (90%) axoglial units where only MDMs were present.

“We found 2–3 MDMs attached to each of the 18/20 (90%) axoglial units where only MDMs were present (Fig. 2).”

Blue group genes up-regulated at recovery included heterogeneous cytokines (IFN-α, IFN-β, TGFB3, IL2, IL3, IL4, IL12α, IL12β, PDGFA, CSF2, and CXCL2).

“Blue group genes up-regulated at recovery included heterogeneous cytokines (IFN-α, IFN-β, TGFB3, IL2, IL3, IL4, IL12α, IL12β, PDGFA, CSF2, and CXCL2).”

More than half of all analyzed MDM and MiDM cells (n = 255 total) contained myelin debris, regardless of whether axon segments were intact or demyelinated.

“More than half of all analyzed MDM and MiDM cells (n = 255 total) contained myelin debris, regardless of whether axon segments were intact or demyelinated (Fig. 2).”

7/75 (9%) of axoglial units demonstrated MDMs attached to nodes of Ranvier.

“Unexpectedly, 7/75 (9%) of axoglial units demonstrated MDMs attached to nodes of Ranvier.”

A subset of MDM-enriched genes were dynamically regulated in monocytes and MDMs but not in microglia.

“Conversely, a subset of MDM-enriched genes were dynamically regulated in monocytes and MDMs but not in microglia (Fig. 6, A and B).”

Equivalent magnitude of weight loss in Ccr2rfp/+::Cx3cr1gfp/+ and Ccr2rfp/rfp::Cx3cr1gfp/+ mice at preonset and onset stages of EAE showed that CCR2 deficiency did not affect systemic inflammation in this model.

“We observed equivalent magnitude of weight loss in Ccr2rfp/+::Cx3cr1gfp/+ and Ccr2rfp/rfp::Cx3cr1gfp/+ mice at preonset and onset stages of EAE, showing that CCR2 deficiency did not affect systemic inflammation in this model (Fig. 5 A).”

Demyelination was significantly reduced at EAE onset in CCR2-deficient mice.

“Demyelination was significantly reduced at EAE onset in CCR2-deficient mice (Fig. 5 F), indicating the importance of MDM recognition of disrupted nodes for efficient inflammatory demyelination.”

MDMs were more likely to express effector functions, including secreted factors and surface molecules (18/28; 64.3% of MDM-enriched genes encoded effector functions).

“Comparing MDM-enriched genes with MiDM-enriched genes showed that MDMs were more likely to express effector functions, including secreted factors and surface molecules (18/28; 64.3% of MDM-enriched genes encoded effector functions; Fig. 6 C; and Table S1, purple genes).”

MDMs, but not MiDMs, frequently contained osmiophilic granules and microvilli.

“MDMs, but not MiDMs, frequently contained osmiophilic granules and microvilli (unpublished data).”

There were unequal numbers of MiDMs and MDMs before and after disease onset.

“Therefore, there were unequal numbers of MiDMs/MDMs before and after disease onset (Fig. 1 B).”

MDMs and MiDMs can be distinguished by cell volume and primary processes.

“MDMs and MiDMs can be distinguished by cell volume and primary processes.”

Most (55/75; 73%) axoglial units are contacted in limited fashion by MDMs and MiDMs.

“Most (55/75; 73%) axoglial units are contacted in limited fashion by MDMs and MiDMs.”

Using criteria detected in confocal analysis, it is possible to distinguish MDMs and MiDMs in SBF-SEM images.

“Using criteria detected in the previous step, it is possible to distinguish MDMs and MiDMs in SBF-SEM images.”

An intratubal MDM with myelin debris interposed between compact myelin and axolemma near a node of Ranvier was detected.

“Further, we detected an intratubal (Stoll et al., 1989) MDMs with myelin debris interposed between compact myelin and axolemma near a node of Ranvier (Fig. 4 B).”

• cites Stoll et al., 1989 sourceNot checked — no full text of the source

MDMs had shorter, thicker mitochondria than MiDMs.

“MDMs had shorter, thicker mitochondria than MiDMs (unpublished data).”

No MiDMs contacted nodes of Ranvier.

“No MiDMs contacted nodes of Ranvier.”

MDMs and MiDMs show characteristic ultrastructural differences regarding their mitochondria, nuclei, osmiophilic granules and microvilli.

“MDMs and MiDMs show characteristic ultrastructural differences regarding their mitochondria, nuclei, osmiophilic granules and microvilli.”

MDMs frequently exhibited morphological characteristics suggesting an involvement in active demyelination.

“MDMs frequently exhibited morphological characteristics suggesting an involvement in active demyelination.”

A subset of genes were expressed in microglia and highly regulated in MiDMs during EAE, but not expressed at all in monocytes or MDMs.

“We noted a subset of genes that were expressed in microglia and highly regulated in MiDMs during EAE, but not expressed at all in monocytes or MDMs (Fig. 6, A and B).”

One representative monocyte surrounded a node of Ranvier with two microvilli interposed between myelin and axolemma near the paranode complex.

“One representative monocyte surrounded a node of Ranvier with two microvilli interposed between myelin and axolemma near the paranode complex (Fig. 4 A).”

A substantial minority of genes both for MDMs and MiDMs showed some dissonant time points, at which a previously down-regulated gene might show up-regulation.

“However, a substantial minority of genes both for MDMs and MiDMs showed some dissonant time points, at which a previously down-regulated gene might show up-regulation (unpublished data).”

MDMs showed remarkably invasive behavior, including extension of microvilli or localization of cell soma between axolemma and myelin sheath.

“showed remarkably invasive behavior, including extension of microvilli (Fig. 4 A) or localization of cell soma (Fig. 4 B) between axolemma and myelin sheath”

The authors found strikingly divergent relationships of MDMs and MiDMs to axoglial units, by quantitative and qualitative ultrastructural analysis.

“Instead, we found strikingly divergent relationships of MDMs and MiDMs to axoglial units, by quantitative and qualitative ultrastructural analysis.”

Expression profiles showed consonant changes for the vast majority of genes analyzed: if a gene was up-regulated at any time point, then its expression level showed an increase at other time points as well.

“These expression profiles showed consonant changes for the vast majority of genes analyzed: if a gene was up-regulated at any time point, then its expression level showed an increase at other time points as well.”

The extent of gene up-regulation in MDMs exceeded that seen in MiDMs.

“Additionally, the extent of gene up-regulation in MDMs exceeded that seen in MiDMs.”

In the Ingenuity IPA database, the terms cell movement, chemoattraction, and migration indicated production of chemokines such as CCL2, CCL3, CCL4, CCL5, and CCL7, which are up-regulated at onset and further increased at peak.

“In the Ingenuity IPA database, the terms cell movement, chemoattraction, and migration indicated production of chemokines such as CCL2, CCL3, CCL4, CCL5, and CCL7, which are up-regulated at onset and further increased at peak (Fig. 6, E and F, green group and genes).”

No functions were reported to be down-regulated in MDMs at EAE onset as compared with naive monocytes.

“No functions were reported to be down-regulated in MDMs at EAE onset as compared with naive monocytes.”

MDMs were unexpectedly observed at nodes of Ranvier in 9% of axoglial units.

“MDMs were unexpectedly observed at nodes of Ranvier in 9% of axoglial units”

MDMs showed up-regulation of functions associated to macrophages, including phagocytosis, calcium signaling, production of prostanoids, adhesion, autophagy, and cell clearance.

“MDMs showed up-regulation of functions associated to macrophages, including phagocytosis, calcium signaling, production of prostanoids, adhesion, autophagy, and cell clearance (Fig. 7 B).”

Grouping of regulated genes into functional categories demonstrated a remarkable down-regulation of microglial metabolism at the nuclear, cytoplasmic and cytoskeletal levels.

“Grouping of regulated genes into functional categories demonstrated a remarkable down-regulation of microglial metabolism at the nuclear, cytoplasmic and cytoskeletal levels.”

Naive and recovery patterns were similar for both cell types.

“Naive and recovery patterns were similar for both cell types.”

Neutrophils in inflamed CNS of Ccr2rfp/rfp::Cx3cr1gfp/+ mice did not recognize disrupted nodes.

“neutrophils in inflamed CNS of Ccr2rfp/rfp::Cx3cr1gfp/+ mice did not recognize disrupted nodes”

Both MiDMs and MDMs demonstrated this pattern of gene-expression kinetics.

“Both MiDMs (Fig. 8 B) and MDMs (Fig. 8 D) demonstrated this pattern of gene-expression kinetics.”

MiDMs showed highly significant up-regulation of functions associated with cell movement, chemoattraction, and migration.

“MiDMs showed highly significant up-regulation of functions associated with cell movement, chemoattraction, and migration (Fig. 7 B).”

Cell motility pathways were clearly different in MiDMs and MDMs.

“Cell motility pathways were clearly different in MiDMs and MDMs (unpublished data).”

MDMs up-regulated a large suite of inflammation-associated genes at EAE onset, with subsequent regression to the expression phenotype of circulating monocytes.

“MDMs up-regulated a large suite of inflammation-associated genes at EAE onset, with subsequent regression to the expression phenotype of circulating monocytes.”

The authors noted differential nuclear morphology, mitochondrial shape, and osmiophilic granule content between MDMs and MiDMs.

“We also noted differential nuclear morphology, mitochondrial shape, and osmiophilic granule content between MDMs and MiDMs.”

GAPDH was up-regulated in MiDMs at onset.

“GAPDH was up-regulated in MiDMs at onset.”

Gene expression profiling across the time course of EAE yielded five patterns as analyzed by k-means clustering.

“Gene expression profiling across the time course of EAE yielded intriguing kinetics as analyzed by k-means clustering. Five patterns were observed.”

The authors characterized a molecular signature for resident microglia at EAE onset.

“we characterized a molecular signature for resident microglia at EAE onset”

Both HIF-1α and TNF pathways were also differentially regulated in MiDMs and MDMs.

“Both HIF-1α (Fig. 9 A) and TNF pathways (not depicted) were also differentially regulated in MiDMs and MDMs.”

MiDM metabolism was severely down-regulated.

“MiDM metabolism was severely down-regulated”

MiDMs exhibited a repressed metabolic and activation phenotype by comparison to naive microglia, including proliferation, RNA metabolism, cytoskeletal organization, microtubule dynamics, extension of processes, phagocytosis and generation of reactive oxygen species.

“In other respects, MiDMs exhibited a repressed metabolic and activation phenotype by comparison to naive microglia (Fig. 7 B) including proliferation, RNA metabolism, cytoskeletal organization, microtubule dynamics, extension of processes, phagocytosis and generation of reactive oxygen species.”

Type I IFN pathway was regulated virtually identically in MiDMs and MDMs.

“By contrast, type I IFN pathway (Fig. 9 B) was regulated virtually identically in MiDMs and MDMs.”

The presence of myelin debris at the peak of EAE did not discriminate MDMs from MiDMs.

“the presence of myelin debris at the peak of EAE did not discriminate MDMs from MiDMs”

At disease onset, microglia showed drastic down-regulation of the expression profile observed in cells from healthy brain.

“At disease onset, microglia showed drastic down-regulation of the expression profile observed in cells from healthy brain.”

To date, no research techniques have permitted distinction between monocytes and microglia in CNS tissue without irradiation chimerism or parabiosis, techniques that confound interpretation or impose practical limitations.

“To date, no research techniques have permitted distinction between monocytes and microglia in CNS tissue without irradiation chimerism or parabiosis, techniques that confound interpretation or impose practical limitations (Ajami et al., 2007, 2011; Ransohoff, 2007).”

• cites Ajami et al., 2007, 2011Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Ransohoff, 2007 sourceNot checked — no full text of the source

Phagocytic microglia appear relatively inert at disease onset.

“In contrast, phagocytic microglia appear relatively inert at disease onset.”

Monocyte-derived macrophages initiate demyelination, often at nodes of Ranvier.

“Here we provide evidence that MDMs initiate demyelination, often at nodes of Ranvier.”

• Also stated in: § MDMs initiated demyelination at EAE onset · plain

  “MDMs initiated demyelination at EAE onset”

Differential functions of monocyte-derived macrophages and microglia-derived macrophages are relevant for human demyelinating disease.

“Therefore, differential functions of MDMs and MiDMs are relevant for human demyelinating disease.”

In each case, the contact between MDMs and node appeared to be pathogenic.

“In each case, the contact between MDMs and node appeared to be pathogenic.”

As nodal pathology was equivalent in Ccr2rfp/rfp::Cx3cr1gfp/+ and Ccr2rfp/+::Cx3cr1gfp/+ mice at the preonset stage of EAE, inflammatory nodal disruption could be reversible if MDMs were prevented from initiating demyelination at those sites.

“Furthermore, as nodal pathology was equivalent in Ccr2rfp/rfp::Cx3cr1gfp/+ (Fig. 5 D) and Ccr2rfp/+::Cx3cr1gfp/+ mice at the preonset stage of EAE, the results suggested that inflammatory nodal disruption could be reversible if MDMs were prevented from initiating demyelination at those sites.”

These observations suggested that MDMs initiated demyelination at the onset of EAE.

“These observations suggested that MDMs initiated demyelination at the onset of EAE.”

MDM recognition of disrupted nodes is important for efficient inflammatory demyelination.

“Demyelination was significantly reduced at EAE onset in CCR2-deficient mice (Fig. 5 F), indicating the importance of MDM recognition of disrupted nodes for efficient inflammatory demyelination.”

MiDMs and MDMs exhibited markedly distinct expression profiles during EAE.

“These observations indicated that MiDMs and MDMs exhibited markedly distinct expression profiles during EAE.”

Cell growth-related genes expressed at this time point correlated to reported patterns of microglial proliferation during EAE.

“Cell growth–related genes expressed at this time point correlated to reported patterns of microglial proliferation during EAE (Ajami et al., 2011).”

• cites Ajami et al., 2011 sourceNot checked — no full text of the source

This observation suggested that initial MDM-axoglial contacts might occur at nodes of Ranvier.

“This observation suggested that initial MDM–axoglial contacts might occur at nodes of Ranvier.”

Comparison of Ccr2rfp/rfp::Cx3cr1gfp/+ and Ccr2rfp/+::Cx3cr1gfp/+ mice at and before EAE onset emphasized the importance of MDMs for this mechanism of demyelination.

“Comparison of Ccr2rfp/rfp::Cx3cr1gfp/+ and Ccr2rfp/+::Cx3cr1gfp/+ mice at and before EAE onset emphasized the importance of MDMs for this mechanism of demyelination.”

This pathway analysis corresponded well to effector properties displayed by MDMs in the SBF-SEM analysis.

“This pathway analysis corresponded well to effector properties displayed by MDMs in our SBF-SEM analysis (Figs. 2–4).”

In this study, the authors developed a novel strategy to discriminate MDMs from MiDMs.

“In this study, we developed a novel strategy to discriminate MDMs from MiDMs.”

The authors' detection of MDMs at nodes of Ranvier is consistent with recent reports of nodal pathology in clinical demyelinated tissues.

“our detection of MDMs at nodes of Ranvier is consistent with recent reports of nodal pathology in clinical demyelinated tissues (Fu et al., 2011; Desmazières et al., 2012)”

• cites Fu et al., 2011Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Desmazières et al., 2012 sourceNot checked — no full text of the source

Expression profiles of MDMs reflected the activated phagocytic phenotype observed through SBF-SEM.

“expression profiles of MDMs reflected the activated phagocytic phenotype observed through SBF-SEM”

Blood monocytes and resident microglia were exposed to the same inflammatory environment.

“Blood monocytes and resident microglia were exposed to the same inflammatory environment.”

• AssumptionRests on an assumption: The EAE disease model exposes both recruited monocytes and resident microglia to the same intensely inflammatory environment.

MiDMs and MDMs have disparate responses to the inflammatory CNS microenvironment of EAE, despite their being present in close proximity.

“This observation underscored disparate responses of MiDMs and MDMs to the inflammatory CNS microenvironment of EAE, despite their being present in close proximity (Fig. 1 A).”

At the onset of EAE, the authors are able to make an unequivocal distinction between resident microglia (CX3CR1gfp) and infiltrating monocytes (CCR2rfp).

“At the onset of EAE, the time point at which our imaging studies were focused, we are able to make an unequivocal distinction between resident microglia (CX3CR1gfp) and infiltrating monocytes (CCR2rfp).”

Gene expression by morphologically identical microglia can differ drastically contingent on context.

“gene expression by morphologically identical microglia can differ drastically contingent on context (Perry et al., 2007).”

• cites Perry et al., 2007 sourceNot checked — no full text of the source

The observed frequency of MDM-nodal interaction represents a minimum estimate as MDMs found at heminodes adjacent to a demyelinated segment were not scored.

“Our observed frequency of MDM–nodal interaction represents a minimum estimate as MDMs found at heminodes adjacent to a demyelinated segment (Fig. 3 B) were not scored.”

MDMs initiate demyelination at EAE onset.

“we showed that MDMs initiate demyelination at EAE onset”

Selection of the EAE disease model ensured that both recruited monocytes and resident microglia were exposed to the same intensely inflammatory environment to increase the likelihood that ambient conditions could activate these two myeloid cell types toward a convergent inflammatory phenotype.

“Selection of the EAE disease model ensured that both recruited monocytes and resident microglia were exposed to the same intensely inflammatory environment to increase the likelihood that ambient conditions could activate these two myeloid cell types toward a convergent inflammatory phenotype.”

• AssumptionThe authors state this as an assumption: Exposure of monocytes and microglia to the same intensely inflammatory environment increases the likelihood that they adopt a convergent inflammatory phenotype.

HIF-1α and TNF signaling may partly drive pathogenic properties of MDMs.

“Collectively, these data suggest that HIF-1α and TNF signaling may partly drive pathogenic properties of MDMs.”

The authors' findings carry biological and medical significance by demonstrating and characterizing differential responses of infiltrating monocytes and resident microglia in a relevant disease model at a prespecified time point, at which point pathogenic events are taking place.

“Our findings carry biological and medical significance by demonstrating and characterizing differential responses of infiltrating monocytes and resident microglia in a relevant disease model at a prespecified time point, at which point pathogenic events are taking place.”