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Pathologic and Protective Roles for Microglial Subsets and Bone Marrow- and Blood-Derived Myeloid Cells in Central Nervous System Inflammation.

Wlodarczyk A, Cédile O, Jensen KN, Jasson A, Mony JT, Khorooshi R, Owens T - Front Immunol (2015)

Bottom Line: However, as in other tissues, neuroinflammation can have beneficial as well as pathological outcomes.Moreover, in contrast to BMDM/DC, both subsets of microglia express protective interferon-beta (IFNβ), high levels of colony-stimulating factor-1 receptor, and do not express the Th1-associated transcription factor T-bet.Taken together, our data suggest that CD11c(+) microglia, CD11c(-) microglia, and infiltrating BMDM/DC represent separate and distinct populations and illustrate the heterogeneity of the CNS inflammatory environment.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology Research, Institute for Molecular Medicine, University of Southern Denmark , Odense , Denmark.

ABSTRACT
Inflammation is a series of processes designed for eventual clearance of pathogens and repair of damaged tissue. In the context of autoimmune recognition, inflammatory processes are usually considered to be pathological. This is also true for inflammatory responses in the central nervous system (CNS). However, as in other tissues, neuroinflammation can have beneficial as well as pathological outcomes. The complex role of encephalitogenic T cells in multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE) may derive from heterogeneity of the myeloid cells with which these T cells interact within the CNS. Myeloid cells, including resident microglia and infiltrating bone marrow-derived cells, such as dendritic cells (DC) and monocytes/macrophages [bone marrow-derived macrophages (BMDM)], are highly heterogeneous populations that may be involved in neurotoxicity and also immunoregulation and regenerative processes. Better understanding and characterization of myeloid cell heterogeneity is essential for future development of treatments controlling inflammation and inducing neuroprotection and neuroregeneration in diseased CNS. Here, we describe and compare three populations of myeloid cells: CD11c(+) microglia, CD11c(-) microglia, and CD11c(+) blood-derived cells in terms of their pathological versus protective functions in the CNS of mice with EAE. Our data show that CNS-resident microglia include functionally distinct subsets that can be distinguished by their expression of CD11c. These subsets differ in their expression of Arg-1, YM1, iNOS, IL-10, and IGF-1. Moreover, in contrast to BMDM/DC, both subsets of microglia express protective interferon-beta (IFNβ), high levels of colony-stimulating factor-1 receptor, and do not express the Th1-associated transcription factor T-bet. Taken together, our data suggest that CD11c(+) microglia, CD11c(-) microglia, and infiltrating BMDM/DC represent separate and distinct populations and illustrate the heterogeneity of the CNS inflammatory environment.

No MeSH data available.


Related in: MedlinePlus

Gating strategy for flow cytometry and FACS-sorting. Representative flow cytometry profiles from individual central nervous system suspensions prepared from mice with severe EAE, showing gating strategy used for flow cytometry and FACS-sorting. First doublets were excluded from live gate (A) based on FSC-H, FSC-W (B) and SSC-H, SSC-W (C), then microglia were gated based on CD45 and CD11b expression (D). CD11c− microglia, CD11c+ microglia, and BMDM/DC population were discriminated based on CD45 and CD11c expression and gated based on isotype control antibodies (vertical line) and sorted (E).
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Figure 1: Gating strategy for flow cytometry and FACS-sorting. Representative flow cytometry profiles from individual central nervous system suspensions prepared from mice with severe EAE, showing gating strategy used for flow cytometry and FACS-sorting. First doublets were excluded from live gate (A) based on FSC-H, FSC-W (B) and SSC-H, SSC-W (C), then microglia were gated based on CD45 and CD11b expression (D). CD11c− microglia, CD11c+ microglia, and BMDM/DC population were discriminated based on CD45 and CD11c expression and gated based on isotype control antibodies (vertical line) and sorted (E).

Mentions: Mice were anesthetized with 0.2 mg pentobarbital (200 mg/ml; Glostrup Apotek, Glostrup, Denmark) per gram body weight and intracardially perfused with ice-cold PBS. CNS tissue (Brain and SC from EAE model, Ipsi lateral part of the brain from NMO-like disease, and whole brain from Cuprizone-induced demyelination) was collected and a single cell suspension was generated by forcing through a 70 μm cell strainer (BD Biosciences). Mononuclear cells were collected after centrifugation on 37% Percoll (GE Healthcare Bio-sciences AB, Brøndby, Denmark). Spleens were digested with collagenase D (0.5 mg/ml; Roche, Hvidovre-Copenhagen) and DNase I (40 μg/ml, Roche) for 30 min at 37°C. Supernatant was collected and supplemented with 100 mM EDTA for 5 min at 37°C and passed through a 70 μm cell strainer (BD Biosciences, Albertslund, Denmark). Red blood cells were lysed with a 0.83% NH4Cl solution. Splenocytes and CNS mononuclear cells were then incubated with anti-Fc receptor (Clone 2.4G2; 1 μg/ml; BD Pharmingen) and Syrian hamster IgG (50 μg/ml; Jackson Immuno Research Laboratories Inc., Skanderborg, Denmark) in PBS 2% fetal bovine serum (FBS). CNS mononuclear cells were stained with PE-anti-mouse CD45 (Biolegend, Copenhagen, Denmark), PerCP-Cy5.5-anti-mouse CD11b (Biolegend) and biotinylated-anti-mouse CD11c (BD Pharmingen) antibodies in PBS 2% FBS. Splenocytes were stained with PE-anti-mouse CD11c antibodies in PBS 2% FBS and CD11c+ cells were sorted on a FACSAria™ III cell sorter (BD Biosciences, Albertslund, Denmark). After excluding doublets (FSC-H, FSC-W and SSC-H, SSC-W) (Figures 1A–C), CNS cell populations were gated based on isotype control antibodies as CD45dimCD11b+CD11c− (CD11c− microglia), CD45dimCD11b+CD11c+ (CD11c+ microglia), and CD45highCD11c+ (BMDM/DC) (Figures 1D,E) and were sorted on a FACSAria™ III cell sorter (BD Biosciences).


Pathologic and Protective Roles for Microglial Subsets and Bone Marrow- and Blood-Derived Myeloid Cells in Central Nervous System Inflammation.

Wlodarczyk A, Cédile O, Jensen KN, Jasson A, Mony JT, Khorooshi R, Owens T - Front Immunol (2015)

Gating strategy for flow cytometry and FACS-sorting. Representative flow cytometry profiles from individual central nervous system suspensions prepared from mice with severe EAE, showing gating strategy used for flow cytometry and FACS-sorting. First doublets were excluded from live gate (A) based on FSC-H, FSC-W (B) and SSC-H, SSC-W (C), then microglia were gated based on CD45 and CD11b expression (D). CD11c− microglia, CD11c+ microglia, and BMDM/DC population were discriminated based on CD45 and CD11c expression and gated based on isotype control antibodies (vertical line) and sorted (E).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4562247&req=5

Figure 1: Gating strategy for flow cytometry and FACS-sorting. Representative flow cytometry profiles from individual central nervous system suspensions prepared from mice with severe EAE, showing gating strategy used for flow cytometry and FACS-sorting. First doublets were excluded from live gate (A) based on FSC-H, FSC-W (B) and SSC-H, SSC-W (C), then microglia were gated based on CD45 and CD11b expression (D). CD11c− microglia, CD11c+ microglia, and BMDM/DC population were discriminated based on CD45 and CD11c expression and gated based on isotype control antibodies (vertical line) and sorted (E).
Mentions: Mice were anesthetized with 0.2 mg pentobarbital (200 mg/ml; Glostrup Apotek, Glostrup, Denmark) per gram body weight and intracardially perfused with ice-cold PBS. CNS tissue (Brain and SC from EAE model, Ipsi lateral part of the brain from NMO-like disease, and whole brain from Cuprizone-induced demyelination) was collected and a single cell suspension was generated by forcing through a 70 μm cell strainer (BD Biosciences). Mononuclear cells were collected after centrifugation on 37% Percoll (GE Healthcare Bio-sciences AB, Brøndby, Denmark). Spleens were digested with collagenase D (0.5 mg/ml; Roche, Hvidovre-Copenhagen) and DNase I (40 μg/ml, Roche) for 30 min at 37°C. Supernatant was collected and supplemented with 100 mM EDTA for 5 min at 37°C and passed through a 70 μm cell strainer (BD Biosciences, Albertslund, Denmark). Red blood cells were lysed with a 0.83% NH4Cl solution. Splenocytes and CNS mononuclear cells were then incubated with anti-Fc receptor (Clone 2.4G2; 1 μg/ml; BD Pharmingen) and Syrian hamster IgG (50 μg/ml; Jackson Immuno Research Laboratories Inc., Skanderborg, Denmark) in PBS 2% fetal bovine serum (FBS). CNS mononuclear cells were stained with PE-anti-mouse CD45 (Biolegend, Copenhagen, Denmark), PerCP-Cy5.5-anti-mouse CD11b (Biolegend) and biotinylated-anti-mouse CD11c (BD Pharmingen) antibodies in PBS 2% FBS. Splenocytes were stained with PE-anti-mouse CD11c antibodies in PBS 2% FBS and CD11c+ cells were sorted on a FACSAria™ III cell sorter (BD Biosciences, Albertslund, Denmark). After excluding doublets (FSC-H, FSC-W and SSC-H, SSC-W) (Figures 1A–C), CNS cell populations were gated based on isotype control antibodies as CD45dimCD11b+CD11c− (CD11c− microglia), CD45dimCD11b+CD11c+ (CD11c+ microglia), and CD45highCD11c+ (BMDM/DC) (Figures 1D,E) and were sorted on a FACSAria™ III cell sorter (BD Biosciences).

Bottom Line: However, as in other tissues, neuroinflammation can have beneficial as well as pathological outcomes.Moreover, in contrast to BMDM/DC, both subsets of microglia express protective interferon-beta (IFNβ), high levels of colony-stimulating factor-1 receptor, and do not express the Th1-associated transcription factor T-bet.Taken together, our data suggest that CD11c(+) microglia, CD11c(-) microglia, and infiltrating BMDM/DC represent separate and distinct populations and illustrate the heterogeneity of the CNS inflammatory environment.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology Research, Institute for Molecular Medicine, University of Southern Denmark , Odense , Denmark.

ABSTRACT
Inflammation is a series of processes designed for eventual clearance of pathogens and repair of damaged tissue. In the context of autoimmune recognition, inflammatory processes are usually considered to be pathological. This is also true for inflammatory responses in the central nervous system (CNS). However, as in other tissues, neuroinflammation can have beneficial as well as pathological outcomes. The complex role of encephalitogenic T cells in multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE) may derive from heterogeneity of the myeloid cells with which these T cells interact within the CNS. Myeloid cells, including resident microglia and infiltrating bone marrow-derived cells, such as dendritic cells (DC) and monocytes/macrophages [bone marrow-derived macrophages (BMDM)], are highly heterogeneous populations that may be involved in neurotoxicity and also immunoregulation and regenerative processes. Better understanding and characterization of myeloid cell heterogeneity is essential for future development of treatments controlling inflammation and inducing neuroprotection and neuroregeneration in diseased CNS. Here, we describe and compare three populations of myeloid cells: CD11c(+) microglia, CD11c(-) microglia, and CD11c(+) blood-derived cells in terms of their pathological versus protective functions in the CNS of mice with EAE. Our data show that CNS-resident microglia include functionally distinct subsets that can be distinguished by their expression of CD11c. These subsets differ in their expression of Arg-1, YM1, iNOS, IL-10, and IGF-1. Moreover, in contrast to BMDM/DC, both subsets of microglia express protective interferon-beta (IFNβ), high levels of colony-stimulating factor-1 receptor, and do not express the Th1-associated transcription factor T-bet. Taken together, our data suggest that CD11c(+) microglia, CD11c(-) microglia, and infiltrating BMDM/DC represent separate and distinct populations and illustrate the heterogeneity of the CNS inflammatory environment.

No MeSH data available.


Related in: MedlinePlus