Resident Mononuclear Phagocytes Research Articles

Macrophages and fibrosis: how resident and infiltrating mononuclear phagocytes account for organ injury, regeneration or atrophy.

Mononuclear phagocytes (MP), i.e., monocytes, macrophages, and dendritic cells (DCs), are essential for immune homeostasis via their capacities to clear pathogens, pathogen components, and non-infectious particles. However, tissue injury-related changes in local microenvironments activate resident and infiltrating MP towards pro-inflammatory phenotypes that contribute to inflammation by secreting additional inflammatory mediators. Efficient control of injurious factors leads to a switch of MP phenotype, which changes the microenvironment towards the resolution of inflammation. In the same way, MP endorses adaptive structural responses leading to either compensatory hypertrophy of surviving cells, tissue regeneration from local tissue progenitor cells, or tissue fibrosis and atrophy. Under certain circumstances, MP contribute to the reversal of tissue fibrosis by clearance of the extracellular matrix. Here we give an update on the tissue microenvironment-related factors that, upon tissue injury, instruct resident and infiltrating MP how to support host defense and recover tissue function and integrity. We propose that MP are not intrinsically active drivers of organ injury and dysfunction but dynamic amplifiers (and biomarkers) of specific tissue microenvironments that vary across spatial and temporal contexts. Therefore, MP receptors are frequently redundant and suboptimal targets for specific therapeutic interventions compared to molecular targets upstream in adaptive humoral or cellular stress response pathways that influence tissue milieus at a contextual level.

Three-Dimensional Visualization With Tissue Clearing Uncovers Dynamic Alterations of Renal Resident Mononuclear Phagocytes After Acute Kidney Injury.

Traditional histologic methods are limited in detecting dynamic changes in immune cells during acute kidney injury (AKI). Recently, optical tissue clearing combined with multiphoton microscopy (MPM) or light sheet fluorescence microscopy (LSFM) has become an emerging method for deep tissue evaluation and three-dimensional visualization. These new approaches have helped expand our understanding of tissue injury and repair processes, including tracing the changes in immune cells. We designed this study to investigate the morphological and functional alterations of renal mononuclear phagocytes (MNPs) in lipopolysaccharide (LPS)-induced AKI using renal clearing in CD11c-YFP mice. We also evaluated the effect of the NLRP3 inhibitor MCC950 to determine whether NLRP3 inhibition attenuates the activation of CD11c+ cells in an LPS-induced AKI model. Transverse sectioned whole mouse kidney imaging by LSFM showed that CD11c+ cells were mainly distributed in the cortex, especially the tubulointerstitial area. The number of CD11c+ cells was significantly more densely interspersed, particularly in periglomerular and perivascular lesions, in the saline-treated LPS-exposed kidney than in the control kidney. Deep imaging of the kidney cortex by MPM demonstrated an increased number of CD11c+ cells in the saline-treated LPS group compared with the control group. This quantitative alteration of CD11c+ cells in AKI was accompanied by morphological changes at high resolution, showing an increased number and level of dendrites. These morphological and behavioral changes in the saline-treated LPS group were accompanied by increased MHC class II and CD86 on CD11c-YFP+ cells. MCC950 attenuated the activation of CD11c+ cells after AKI and improved renal function. In conclusion, wide and deep three-dimensional visualization using MPM or LSFM combined with kidney clearing uncovers dynamic changes of renal MNPs, which are directly linked to renal function in AKI.

Early antitumor activity of oral Langerhans cells is compromised by a carcinogen

Early diagnosis of oral squamous cell carcinoma (OSCC) remains an unmet clinical need. Therefore, elucidating the initial events of OSCC preceding tumor development could benefit OSCC prognosis. Here, we define the Langerhans cells (LCs) of the tongue and demonstrate that LCs protect the epithelium from carcinogen-induced OSCC by rapidly priming αβT cells capable of eliminating γH2AX+ epithelial cells, whereas γδT and natural killer cells are dispensable. The carcinogen, however, dysregulates the epithelial resident mononuclear phagocytes, reducing LC frequencies, while dendritic cells (DCs), macrophages, and plasmacytoid DCs (pDCs) populate the epithelium. Single-cell RNA-sequencing analysis indicates that these newly differentiated cells display an immunosuppressive phenotype accompanied by an expansion of T regulatory (Treg) cells. Accumulation of the Treg cells was regulated, in part, by pDCs and precedes the formation of visible tumors. This suggests LCs play an early protective role during OSCC, yet the capacity of the carcinogen to dysregulate the differentiation of mononuclear phagocytes facilitates oral carcinogenesis.

Importance of particle size of oligomannose-coated liposomes for induction of Th1 immunity

Oligomannose-coated liposomes (OMLs) comprised of dipalmitoylphosphatidylcholine,cholesterol and Man3-DPPE at a molar ratio of 1:1:0.1 and particle diameters of about 1000 nm can induce liposome-encased antigen-specific strong Th1 immunity. In this study, we evaluated the effect of particle sizes of OMLs on induction of Th1 immune responses in mice. Spleen cells obtained from mice immunized with antigen-encapsulating OMLs with 1000- and 800-nm diameters secreted remarkably high levels of IFN-γ upon in vitro stimulation. In addition, sera of mice that received these OMLs had significantly higher titers of antigen-specific IgG2a than those of IgG1, which are commonly associated with Th1 responses. In contrast, treatment with antigen-encapsulating OMLs with 400- and 200-nm diameters failed to induce IFN-γ secretion from spleen cells, although these OMLs did elicit elevation of antigen-specific IgGs. In addition, the titers of serum antigen-specific IgG2a were the same as those of IgG1 in mice that received 400-nm OMLs. Resident peritoneal mononuclear phagocytes (MNPs) treated with OMLs of diameter ≥ 600 nm secreted IL-12, which is essential for induction of Th1 immune responses, while those treated with OMLs of ≤ 400 nm failed to produce this cytokine. However, 400-nm OMLs did induce enhanced expression of MHC class II and costimulatory molecules on MNPs, similarly to OMLs of ≥ 600 nm. Taken together, these results strongly indicate that OMLs of diameter ≥ 600 nm are required to induce Th1 immune responses against OML-encased antigens, although OMLs of diameter ≤ 400 nm can activate MNPs.

Incretin drugs in diabetic kidney disease: biological mechanisms and clinical evidence.

As the prevalence of diabetes continues to climb, the number of individuals living with diabetic complications will reach an unprecedented magnitude. The emergence of new glucose-lowering agents - sodium-glucose cotransporter 2 inhibitors and incretin therapies - has markedly changed the treatment landscape of type 2 diabetes mellitus. In addition to effectively lowering glucose, incretin drugs, which include glucagon-like peptide 1 receptor (GLP1R) agonists and dipeptidyl peptidase 4 (DPP4) inhibitors, can also reduce blood pressure, body weight, the risk of developing or worsening chronic kidney disease and/or atherosclerotic cardiovascular events, and the risk of death. Although kidney disease events have thus far been secondary outcomes in clinical trials, an ongoing phase III trial in patients with diabetic kidney disease will test the effect of a GLP1R agonist on a primary kidney disease outcome. Experimental data have identified the modulation of innate immunity and inflammation as plausible biological mechanisms underpinning the kidney-protective effects of incretin-based agents. These drugs block the mechanisms involved in the pathogenesis of kidney damage, including the activation of resident mononuclear phagocytes, tissue infiltration by non-resident inflammatory cells, and the production of pro-inflammatory cytokines and adhesion molecules. GLP1R agonists and DPP4 inhibitors might also attenuate oxidative stress, fibrosis and cellular apoptosis in the kidney.

Importance of methodology in the evaluation of renal mononuclear phagocytes and analysis of a model of experimental nephritis with Shp1 conditional knockout mice

Tissue resident mononuclear phagocytes (Mophs), comprising monocytes, macrophages, and dendritic cells (DCs), play important roles under physiological and pathological conditions. The presence of these cells in the kidney has been known for decades, and studies of renal Mophs (rMophs) are currently underway. Since no unified procedure has been identified to isolate rMophs, results of flow cytometric analysis of rMophs have been inconsistent among studies. We therefore first evaluated a preparative method for rMophs using collagenous digestion. The yield of rMophs greatly increased after the collagenase digestion. In particular, F4/80high rMophs, which were positive for CD11c, a specific marker of DCs, dramatically increased. In addition, since neutrophils are sometimes mixed among rMophs in the analysis of flow cytometry, we established a gating strategy for eliminating neutrophils. To determine the contribution of rMophs to the development of autoimmune nephritis, we analyzed an experimental model of autoimmune nephritis that was applied to Shp1 conditional knockout mice (Shp1 CKO). This knockout strain is generated by crossing a mouse line carrying floxed Shp1 allele to mice expressing Cre recombinase under the control of the CD11c promoter. Shp1 CKO therefore specifically lack Shp1 in cells expressing CD11c. As a result, Shp1 CKO were susceptible to that experimental glomerulonephritis and F4/80high rMophs of Shp1 CKO increased dramatically. In conclusion, our preparative methods for collagenase digestion and gating strategy for neutrophils are necessary for the analysis of rMophs, and Shp1 suppresses the development of autoimmune nephritis through the control of rMophs.

The role of the immune system in driving neuroinflammation.

Neuroinflammation is now recognised as an important contributory factor in the progression of Alzheimer’s disease and probably also in the early stages of the disease. It is likely that this derives largely from aberrant activation of microglia, the resident mononuclear phagocytes of the brain. These cells are responsible for physiological immune surveillance and clearance of pathogens in the central nervous system, but evidence indicates that in Alzheimer’s disease, microglial function is compromised, and this contributes to the pathology. It is unclear what factors cause the inappropriate activation of the microglia in Alzheimer’s disease, but one contributor may be infiltrating peripheral immune cells and these include macrophages and T cells. It has been suggested that both cell types modulate the phenotype of microglia, highlighting the importance of crosstalk between the innate and adaptive immune system in Alzheimer’s disease. This review outlines our current knowledge of how cells of the peripheral immune system, specifically macrophages and T cells, may modulate microglial phenotype in the context of Alzheimer’s disease and considers the impact on their function, especially phagocytic capacity.

High Resolution Intravital Imaging of the Renal Immune Response to Injury and Infection in Mice.

We developed an experimental set up that enables longitudinal studies of immune cell behavior in situ in the challenged as well as unchallenged kidney of anesthetized mice over several hours. Using highly controlled vacuum to stabilize the kidney, the superficial renal cortex could continuously be visualized with minimal disruption of the local microenvironment. No visible changes in blood flow or neutrophils and macrophages numbers were observed after several hours of visualizing the unchallenged kidney, indicating a stable tissue preparation without apparent tissue damage. Applying this set up to monocyte/macrophage (CX3CR1GFP/+) reporter mice, we observed the extensive network of stellate-shaped CX3CR1 positive cells (previously identified as renal mononuclear phagocytes). The extended dendrites of the CX3CR1 positive cells were found to bridge multiple capillaries and tubules and were constantly moving. Light induced sterile tissue injury resulted in rapid neutrophil accumulation to the site of injury. Similarly, microinfusion of uropathogenic Escherichia coli into a single nephron induced a rapid and massive recruitment of neutrophils to the site of infection, in addition to active bacterial clearance by neutrophils. In contrast, the kidney resident mononuclear phagocytes were observed to not increase in numbers or migrate toward the site of injury or infection. In conclusion, this model allows for longitudinal imaging of responses to localized kidney challenges in the mouse.

Fbxw7 increases CCL2/7 in CX3CR1hi macrophages to promote intestinal inflammation.

Resident and inflammatory mononuclear phagocytes (MPh) with functional plasticity in the intestine are critically involved in the pathology of Inflammatory Bowel Diseases (IBD), in which the mechanism remains incompletely understood. In the present study, we found that increased expression of E3 ligase FBXW7 in the inflamed intestine was significantly correlated to IBD severity in both human diseases and mice model. Myeloid-Fbxw7 deficiency protected mice from dextran sodium sulfate (DSS) and 2,6,4-trinitrobenzene sulfonic acid (TNBS) induced colitis. Fbxw7 deficiency resulted in decreased production of chemokines CCL2 and CCL7 by colonic CX3CR1hi resident macrophages and reduced accumulation of CX3CR1int pro-inflammatory MPh in colitis colon tissue. Mice received AAV-shFbxw7 administration showed significantly improved survival rate and alleviated colitis. Mechanisms screening demonstrated that FBXW7 suppresses H3K27me3 modification and promotes Ccl2 and Ccl7 expression via degradation of histone-lysine N-methyltransferase EZH2 in macrophages. Taken together, our results indicate that FBXW7 degrades EZH2 and increases Ccl2/Ccl7 in CX3CR1hi macrophages, which promotes the recruiting CX3CR1int pro-inflammatory MPh into local colon tissues with colitis. Targeting FBXW7 might represent a potential therapeutic approach for intestine inflammation intervention.

Molecular profiling of resident and infiltrating mononuclear phagocytes during rapid adult retinal degeneration using single-cell RNA sequencing

Neuroinflammation commonly accompanies neurodegeneration, but the specific roles of resident and infiltrating immune cells during degeneration remains controversial. Much of the difficulty in assessing myeloid cell-specific functions during disease progression arises from the inability to clearly distinguish between activated microglia and bone marrow-derived monocytes and macrophages in various stages of differentiation and activation within the central nervous system. Using an inducible model of photoreceptor cell death, we investigated the prevalence of infiltrating monocytes and macrophage subpopulations after the initiation of degeneration in the mouse retina. In vivo retinal imaging revealed infiltration of CCR2+ leukocytes across retinal vessels and into the parenchyma within 48 hours of photoreceptor degeneration. Immunohistochemistry and flow cytometry confirmed and characterized these leukocytes as CD11b+CD45+ cells. Single-cell mRNA sequencing of the entire CD11b+CD45+ population revealed the presence of resting microglia, activated microglia, monocytes, and macrophages as well as 12 distinct subpopulations within these four major cell classes. Our results demonstrate a previously immeasurable degree of molecular heterogeneity in the innate immune response to cell-autonomous degeneration within the central nervous system and highlight the necessity of unbiased high-throughput and high-dimensional molecular techniques like scRNAseq to understand the complex and changing landscape of immune responders during disease progression.

Intestinal epithelial cell-specific RARα depletion results in aberrant epithelial cell homeostasis and underdeveloped immune system.

Retinoic acid (RA), a dietary vitamin A metabolite, is crucial in maintaining intestinal homeostasis. RA acts on intestinal leukocytes to modulate their lineage commitment and function. Although the role of RA has been characterized in immune cells, whether intestinal epithelial cells (IECs) rely on RA signaling to exert their immune-regulatory function has not been examined. Here we demonstrate that lack of retinoic acid receptor alpha (RARα) signaling in IECs results in deregulated epithelial lineage specification, leading to increased numbers of goblet cells and Paneth cells. Mechanistically, lack or RARα resulted in increased KLF4+ goblet cell precursors in the distal bowel, whereas RA treatment inhibited klf4 expression and goblet cell differentiation in zebrafish. These changes in secretory cells are associated with increased reg3g, reduced luminal bacterial detection and an underdeveloped intestinal immune system, as evidenced by an almost complete absence of lymphoid follicles and gut resident mononuclear phagocytes. This underdeveloped intestinal immune system shows a decreased ability to clear infection with Citrobacter rodentium. Collectively, our findings indicate that epithelial cell-intrinsic RARα signaling is critical to the global development of the intestinal immune system.

The Role of Lipid Bodies in the Microglial Aging Process and Related Diseases.

Lipid bodies (LBs) have long been considered to be organelles merely for the storage of neutral lipids. However, recent studies have shown the significance of LBs in signal transduction, especially in glial cells, including microglia. Microglial cells are the resident mononuclear phagocytes in the central nervous system and have a close relationship with the aging process and neurodegenerative diseases. Evidence suggests that LBs accumulate and are remodeled during the aging process and the development of neuroinflammatory conditions. However, the mechanisms underlying the formation of LBs under these conditions and the mechanism by which LB remodeling influences the progression of neurodegeneration remain to be clarified. In this review, we have summarized the findings from recent studies with the aim of further elucidating these issues.

Circadian rhythm disruption promotes alcohol-induced liver and brain injury.

Abstract Alcoholic liver disease (ALD) progression to cirrhosis and development of hepatic encephalopathy (HE) are characterized by hepatic and brain inflammation involving innate immune cells. Identifying the risk factors and immune mechanisms involved in susceptibility to liver and brain injury are significant unmet needs. Circadian rhythm disruption promotes alcohol-induced liver pathology. We aim to investigate whether circadian rhythm disruption also primes brain inflammation by impacting trafficking and inflammatory potential of recruited and resident mononuclear phagocytes (MP). Alcohol (EtOH) is delivered to C57BL/6 mice per a modified NIAAA model of alcoholic liver disease whereby chronic alcohol consumption (20% EtOH v/v in drinking water, 4 wks (Meadows-Cook diet)) is combined with three once daily binges of alcohol. Tissue was assessed via H&E and IHC for α-smooth muscle actin (αSMA) and collagen I (Col I) (liver) and allograft inflammatory factor 1 (Iba-1) (brain). All features associated with human alcoholic liver injury including steatosis, hepatic inflammation and early fibrogenesis were present. Brain mononuclear phagocytes analysed by flow cytometry revealed an increase in recruited and resident MP in response to the alcohol treatment. Moreover, in the steady-state, hepatic and brain MP populations demonstrate circadian oscillations. Thus, circadian rhythm disruption may modulate alcohol-induced alterations in mononuclear phagocyte trafficking and function similarly in brain and liver.

Microglia as Potential Regulators of Empathy and Prosocial Behavior – A Hypothesis

Microglia are the primary resident mononuclear phagocytes that maintain central nervous system homeostasis under physiological and pathological conditions. Recent evidence has shown that they differ considerably from other macrophages by contributing to pre- and postnatal brain development by controlling processes such as neurogenesis, oligodendrogenesis, progenitor numbers and the generation and elimination of synapses. Here I Dance round a hypothetical microglial role in modulating empathy and prosocial behavior via influencing the development of associated nervous structures, including the ventromedial and dorsolateral prefrontal cortex, anterior cingulate, precuneus, inferior frontal gyrus, insula and striatum. A potential contributing role of microglia in shaping normal interindividual differences in empathetic abilities and related behaviors is also discussed.

Gimme a complex! Resident mononuclear phagocytes in the kidney as monitors of circulating antigens and immune complexes

Over the past decade, researchers have made substantial progress in characterizing a network of mononuclear phagocytes in the kidney, which variously have been referred to as resident macrophages or dendritic cells. Two recent studies, published in Cell and Kidney International, have identified these resident macrophages/dendritic cells as local immune monitors of peritubular capillaries for circulating antigens and immune complexes. These cells appear to represent an early line of defense against circulating infectious particles and immune complexes, but the resulting inflammatory response may also contribute to interstitial inflammation and kidney disease progression.

Colony stimulating factor-1 receptor as a treatment for cognitive deficits postfractionated whole-brain irradiation.

Whole-brain irradiation (WBI) is commonly used to treat primary tumors of the central nervous systems tumors as well as brain metastases. While this technique has increased survival among brain tumor patients, the side effects of including a decline in cognitive abilities that are generally progressive. In an effort to combat WBI side effects, researchers explored the treatment of colony stimulating factor-1 receptor (CSF-1R) inhibitor. Data show that when a CSF-1R inhibitor is administered with fractionated WBI treatment, there is a decline in the number of resident and peripheral mononuclear phagocytes, a decrease in dendritic spine loss and a reduction in functional and memory deficits. CSFR-1R inhibitors have displayed promising results as an effective counter-treatment for WBI-induced deficits. Further research is required to optimize treatment strategies, establish a treatment timeline and gain a better understanding of the long-term side effects of targeting CSF-1R as a treatment strategy for WBI symptoms. This paper is a review article. Referred literature in this paper has been listed in the references section. The datasets supporting the conclusions of this article are available online by searching various databases, including PubMed. Some original points in this article come from the laboratory practice in our research center and the authors’ experiences.

Interferon-beta signaling in retinal mononuclear phagocytes attenuates pathological neovascularization.

Age‐related macular degeneration (AMD) is a leading cause of vision loss among the elderly. AMD pathogenesis involves chronic activation of the innate immune system including complement factors and microglia/macrophage reactivity in the retina. Here, we show that lack of interferon‐β signaling in the retina accelerates mononuclear phagocyte reactivity and promotes choroidal neovascularization (CNV) in the laser model of neovascular AMD. Complete deletion of interferon‐α/β receptor (Ifnar) using Ifnar1−/− mice significantly enhanced early microglia and macrophage activation in lesion areas. This triggered subsequent vascular leakage and CNV at later stages. Similar findings were obtained in laser‐treated Cx3cr1 Cre ER:Ifnar1 fl/fl animals that allowed the tamoxifen‐induced conditional depletion of Ifnar in resident mononuclear phagocytes only. Conversely, systemic IFN‐β therapy of laser‐treated wild‐type animals effectively attenuated microgliosis and macrophage responses in the early stage of disease and significantly reduced CNV size in the late phase. Our results reveal a protective role of Ifnar signaling in retinal immune homeostasis and highlight a potential use for IFN‐β therapy in the eye to limit chronic inflammation and pathological angiogenesis in AMD.

Abstract 162: Resident Aortic Intimal Mononuclear Phagocytes (MNPs) in the Promotion of Atherosclerosis

Atherosclerosis is an underlying cause of cardiovascular disease (CVD) and a leading cause of morbidity and mortality worldwide. Atherosclerosis promotes CVD through plaque formation, restricted blood flow, and thrombotic events. Macrophage accumulation in plaques, their uptake of cholesterol, and subsequent local death drive disease progression, however, there is growing appreciation for more diverse roles of myeloid cells during disease progression. Here, we focused on characterization of so-called vascular dendritic cells (DCs), which we refer to as aortic mononuclear phagocytes (MNP), that reside under the endothelium in plaque-prone areas. Using en face whole-mount confocal microscopy of aortas, we confirm a uniform resident CD11c+ CX3CR1+ MHCII+ MNP population, even in C57/BL6 mice resistant to atherosclerosis. We find aortic MNPs require M-CSF and Flt3 signaling for survival, but are independent of CCR2 and GM-CSF receptor signaling, making them a distinct myeloid population. They express macrophage-restricted genes LysM and CD64, but not dendritic cell specific genes zBTB46 or L-myc. Lineage-tracing analysis using CD115creER and Flt3cre reporter mice indicate that aortic MNPs likely differentiate from definitive hematopoiesis and not early yolk sac progenitors. Parabiosis experiments show that aortic MNPs are self-maintained independent of blood-born progenitors, failing to exchange with blood progenitors for up to 5 months. Aortic MNPs are different from either typical DCs or macrophages and instead appear to blend distinguishing features of each, more closely resembling macrophages. Using our characterization data, we hope to develop an inducible cell-tracking and a cell-depletion model to differentiate the function of aortic MNPs during the progression of atherosclerosis. Overall, we anticipate these functional analyses will reveal, for the first time, the role of resident aortic MNPs in atherosclerosis.

Serotonin 2B receptor slows disease progression and prevents degeneration of spinal cord mononuclear phagocytes in amyotrophic lateral sclerosis.

Microglia are the resident mononuclear phagocytes of the central nervous system and have been implicated in the pathogenesis of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). During neurodegeneration, microglial activation is accompanied by infiltration of circulating monocytes, leading to production of multiple inflammatory mediators in the spinal cord. Degenerative alterations in mononuclear phagocytes are commonly observed during neurodegenerative diseases, yet little is known concerning the mechanisms leading to their degeneration, or the consequences on disease progression. Here we observed that the serotonin 2B receptor (5-HT2B), a serotonin receptor expressed in microglia, is upregulated in the spinal cord of three different transgenic mouse models of ALS. In mutant SOD1 mice, this upregulation was restricted to cells positive for CD11b, a marker of mononuclear phagocytes. Ablation of 5-HT2B receptor in transgenic ALS mice expressing mutant SOD1 resulted in increased degeneration of mononuclear phagocytes, as evidenced by fragmentation of Iba1-positive cellular processes. This was accompanied by decreased expression of key neuroinflammatory genes but also loss of expression of homeostatic microglial genes. Importantly, the dramatic effect of 5-HT2B receptor ablation on mononuclear phagocytes was associated with acceleration of disease progression. To determine the translational relevance of these results, we studied polymorphisms in the human HTR2B gene, which encodes the 5-HT2B receptor, in a large cohort of ALS patients. In this cohort, the C allele of SNP rs10199752 in HTR2B was associated with longer survival. Moreover, patients carrying one copy of the C allele of SNP rs10199752 showed increased 5-HT2B mRNA in spinal cord and displayed less pronounced degeneration of Iba1 positive cells than patients carrying two copies of the more common A allele. Thus, the 5-HT2B receptor limits degeneration of spinal cord mononuclear phagocytes, most likely microglia, and slows disease progression in ALS. Targeting this receptor might be therapeutically useful.

Validation of Flow Cytometry and Magnetic Bead-Based Methods to Enrich CNS Single Cell Suspensions for Quiescent Microglia.

Microglia are resident mononuclear phagocytes within the CNS parenchyma that intimately interact with neurons and astrocytes to remodel synapses and extracellular matrix. We briefly review studies elucidating the molecular pathways that underlie microglial surveillance, activation, chemotaxis, and phagocytosis; we additionally place these studies in a clinical context. We describe and validate an inexpensive and simple approach to obtain enriched single cell suspensions of quiescent parenchymal and perivascular microglia from the mouse cerebellum and hypothalamus. Following preparation of regional CNS single cell suspensions, we remove myelin debris, and then perform two serial enrichment steps for cells expressing surface CD11b. Myelin depletion and CD11b enrichment are both accomplished using antigen-specific magnetic beads in an automated cell separation system. Flow cytometry of the resultant suspensions shows a significant enrichment for CD11b(+)/CD45(+) cells (perivascular microglia) and CD11b(+)/CD45(-) cells (parenchymal microglia) compared to starting suspensions. Of note, cells from these enriched suspensions minimally express Aif1 (aka Iba1), suggesting that the enrichment process does not evoke significant microglial activation. However, these cells readily respond to a functional challenge (LPS) with significant changes in the expression of molecules specifically associated with microglia. We conclude that methods employing a combination of magnetic-bead based sorting and flow cytometry produce suspensions highly enriched for microglia that are appropriate for a variety of molecular and cellular assays.