Investigating the relationship between Microglia and Monocytes in central nervous system disease

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Microglia and Monocytes bear little reSEMblance in CNS disease

The ability to distinguish between microglia and monocyte cells in CNS tissue via SBF-SEM has enabled a greater understanding of the mechanisms underlying autoimmune pathology. A novel method supports the concept that both cells display differential functional roles in CNS disease.

When an angry mob gather at a scene of impending violence or destruction, an onlooker may find it difficult to distinguish between the individuals who are causing trouble, those who are innocent bystanders and those who may be trying to prevent the ugly proceedings. In many ways, researchers investigating the role of macrophages in autoimmune pathology have faced a similar conundrum with respect to the roles played by microglia- and monocyte-derived macrophages in CNS disease. . In this landmark paper, Yamazaki et al. have shed light on the role of each type of macrophage using serial block face scanning electron microscopy (SBF-SEM) and clearly differentiated MDMs and MiDMs in addition to examining the morphological relationship to axoglia for the very first time in the field.

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Macrophages are leukocytes that digest microbes, foreign substances, cellular debris, and tumour cells via phagocytosis. The two main phenotypes are the classically activated “M1” cells and the alternatively activated “M2” cells. The M1 macrophages/microglia are proinflammatory, being associated with the release of proinflammatory cytokines such as interleukin-1β and tumour necrosis factor-α. M1 cells express CD86 and CD16/32 markers on tyheir cell surfaces and possess inducible nitric oxide synthase activity. Conversely, the M2 macrophages/microglia are anti-inflammatory, associated with the release of anti-inflammatory cytokines such as Interleukin-10. An important characteristic for identification of this phenotype is the expression of CD206 mannose receptor on the surface, and the presence of the enzyme arginase 1.

Most macrophages find themselves positioned at strategic points prone to microbial invasion and accumulation of foreign particles, known as the mononuclear phagocyte system. Microglia are the resident macrophages (MiDM) of the CNS whereas monocyte derived macrophages (MDM), the resident macrophages in blood, provide functional assistance to the former cells1. Distinct ontogenic differences (Ginheaux et al. 2010) and expression profiles (chiu et al. 2013) have heavily implied a difference in functional roles in the pathogenic process. Both macrophages have been heavily implicated in the demyelination process characteristic of autoimmune pathologies such as Multiple Sclerosis (MS) and experimental autoimmune encephalitis (EAE), the animal model of MS, by previous studies4 but little in the way of evidence had hitherto been obtained. In spite of scientists’ valiant efforts to develop techniques for distinguishing the cells in mice, their results had always proved unsatisfactory due to experimental factors that confounded their results, such as irradiation chimerism and parabosis.

The innate immune cells of the brain were first identified nearly a century ago, but only in the past few years have researchers discovered that microglial cells emerge at a different place and time in development than monocytes. Microglia develops from erythromyeloid precursors in the umbilical vesicle, whilst monocytes undergo continuous differentiation throughout postnatal life, from bone marrow hematopoietic stem cells reuiring the Myb transcription factor. Microglial precursors are Myb independent, and microglia themselves self-renew independently of bone marrow haematopoietic stem cells. A few contemporary studies have found microglia have additional properties beyond their roles of macrophages and are crucial for healthy brain function (Olah et al. 2011). MS researchers have thought that the two cells have distinct roles in pathology, owing to their different origins and that although they may look alike, they would not behave alike (Ransohoff, 2014).

MS is an inflammatory disease in which the myelin sheathes, that act as insulating covers for the nerve cells of the brain and spinal cord, become damaged. This results in a disruption of the affected portions of the nervous system ability to communicate, producing a range of symptoms including loss of sensitivity, muscular weakness, muscle spasms and difficulty with co-ordination and movement, amongst other symptoms. It is the most common autoimmune disorder affecting the CNS, with between 2-2.5 million people affected globally as of 20085. Two important characteristics of the disease are considerable macrophage infiltration and prominent activation of resident microglia. Indeed, macrophages represent the principal type of immune cell present in MS autopsy studies and the level of macrophage infiltration has been associated with disease severity.

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The study is an extension of earlier work (Saederup et al., 2010; Mizutani et al., 2012) in which F4/80+ macrophages were isolated from the CNS and flow cytometry was used to analyse cells from double-heterozygous Ccr2rfp::Cx3cr1gfp mice with EAE. In the study GFP was expressed by CD45dim/Ly6C microglia, and RFP was restricted to CD45high/Ly6C+ monocytes. The findings hinted at a possible approach of distinguishing the roles of MiDMs and MDMs in EAE based on the differential expression of GFP and RFP reporters, and provided Yamazaki et al. with a strategy to test the hypothesis regarding differential functional roles of MDMs and MiDMs in neuroinflammation.

The authors of the paper used a recently developed model that tagged the different myeloid cells with different fluorochrome markers and could be used to discriminate between MDMs and MiDMs at the onset of EAE in mice. The histology analysis strategy involved the use of confocal analysis to distinguish MDMs (RFP+) from MiDMs (GFP+) and, using the cell volume and primary process criteria obtained in this step, SBF-SEM was employed to distinguish the two cell types in SBF-SEM images. These images were then inspected to determine ultrastructural differences between MDMs and MiDMs. The relation of MDMs and MiDMs to axoglial units were also quantified and a 3D reconstruction of four representative MDMs at axoglial units was carried out to detect the MDM-axoglial relationship at the onset of EAE.

Immunofluroescent staining at EAE onset found clear morphological differences between MDM and MiDM cells, with a spindle shape being revealed in MDM cells whilst MiDM cells demonstrated more of a process-bearing morphology. MiDM cells were also found to be of much larger size based on the 3D reconstructions using confocal z-stacking imagery. Confocal microscopy identified key ultrastructural differences between the two cell types at EAE onset, with many organelles displaying differentiation such as the nuclei, mitochondria and microvilli. MDMs had shorter, thicker mitochondria than MiDMs, and their nuclei were found to be irregular or bi-lobulated compared to the round nuclei of MiDMs. When taken together, these ultrastructural differences were sufficient to confidently distinguish between MDMs and MiDMs.

The team then looked into the relationship between MDM and MiDM to axoglial units at the onset of EAE, using SFB-SEM. Contacts made by MDM and MiDM with axoglial units were quantified and it was found that most intact and demyelinated axoglial units contacted MDMs and MiDMs. Nearly all axoglial units made contact with MDM when in the presence of one myeloid cell type. Of particular note was that over 90% of MDMs in sole contact with an axon contained myelin, pointing towards the cell potentially being involved in the process of active demyelination. Keen to pursue this line of enquiry, the authors evaluated the MDM-axoglial unit relationship with 3D reconstruction via SFB-SEM, finding morphological characteristics that heavily implicated MDM in active demyelination whereas MiDM cells displayed no such characteristics. Interestingly, Yamazki et al. also found that 9% of axioglial units had MDMs attached to nodes of Ranvier, displaying seemingly pathogenic contact suggesting that initial MDM contact with axoglial units may occur at nodes of Ranvier. No MiDMs had contact with nodes of Ranvier.

To further investigate the role of MDMs play in demyelination at the onset of EAE, the team studied the nodal pathology in Ccr2rfp/rfp::Cx3cr1gfp/+ mice, in which MDMs were mostly absent and largely replaced by neutrophils in inflamed EAE tissue. SFB-SEM was used to examine nodal pathology, myeloid cell-axoglial unit contact and demyelination. Demyelination was significantly reduced at EAE onset in CCR2-defcient mice indicating the importance of MDM recognition of disrupted nodes for efficient inflammatory demyelination,

Expression profiling was used by the researchers to see if the gene expression profiles of MDM and MiDM during the onset of EAE was indicative of different phenotypical and effector properties. An nCounter digital multiplexed gene expression analysis was carried out using ex vivo naive microglia and splenic F4/80+ macrophages as well as MDMs and MiDMs sorted by flow cytometry over the course of EAE onset. It was noted that, over the course of EAE, a subset of genes was expressed in microglia and regulated in MiDMs but not expressed in monocytes or MDMs at all. In reverse, there was also a gene subset regulated in monocytes and MDMs but not in microglia or MiDMs. This supported the team’s hypothesis as the gene expression profiles over the course of EAE were found to be very different.

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Yamazaki et al. also looked into the differential expression of effector functions by MDMs and MiDMs, hoping for some insight into the pathogenesis of the disease. K means clustering was used to discriminate distinct patterns of gene expression in MiDMs over the course of EAE. Five gene groups were identified. Red group genes, which increased in MiDMs at EAE onset consisted mainly of surface proteins. Green group genes, comprised of mostly complement components, chemokines, proliferation genes and inflammation related genes, were up-regulated at EAE onset and furthermore at the peak. Blue group genes, up-regulated at the recovery stage, consisted of heterogeneous cytokines. However, none of the gene groups showed any regulation patterns in MDMs, consistent with the different responses elicited by MDMs and MiDMs over the course of EAE.

Yamazaka et al. have produced a landmark study in the field of autoimmune pathology, furthering our understanding