Juan G. Zarruk

TNF and Increased Intracellular Iron Alter Macrophage Polarization to a Detrimental M1 Phenotype in the Injured Spinal Cord

Macrophages and microglia can be polarized along a continuum toward a detrimental (M1) or a beneficial (M2) state in the injured CNS. Although phagocytosis of myelin in vitro promotes M2 polarization, macrophage/microglia in the injured spinal cord retain a predominantly M1 state that is detrimental to recovery. We have identified two factors that underlie this skewing toward M1 polarization in the injured CNS. We show that TNF prevents phagocytosis-mediated conversion from M1 to M2 cells in vitro and in vivo in spinal cord injury (SCI). Additionally, iron that accumulates in macrophages in SCI increases TNF expression and the appearance of a macrophage population with a proinflammatory mixed M1/M2 phenotype. In addition, transplantation experiments show that increased loading of M2 macrophages with iron induces a rapid switch from M2 to M1 phenotype. The combined effect of this favors predominant and prolonged M1 macrophage polarization that is detrimental to recovery after SCI.

Lipocalin 2 is a novel immune mediator of experimental autoimmune encephalomyelitis pathogenesis and is modulated in multiple sclerosis.

Experimental autoimmune encephalomyelitis (EAE) is a widely used animal model of multiple sclerosis (MS), an inflammatory, demyelinating disease of the central nervous system (CNS). EAE pathogenesis involves various cell types, cytokines, chemokines, and adhesion molecules. Given the complexity of the inflammatory response in EAE, it is likely that many immune mediators still remain to be discovered. To identify novel immune mediators of EAE pathogenesis, we performed an Affymetrix gene array screen on the spinal cords of mice at the onset stage of disease. This screening identified the gene encoding lipocalin 2 (Lcn2) as being significantly upregulated. Lcn2 is a multi‐functional protein that plays a role in glial activation, matrix metalloproteinase (MMP) stabilization, and cellular iron flux. As many of these processes have been implicated in EAE, we characterized the expression and role of Lcn2 in this disease in C57BL/6 mice. We show that Lcn2 is significantly upregulated in the spinal cord throughout EAE and is expressed predominantly by monocytes and reactive astrocytes. The Lcn2 receptor, 24p3R, is also expressed on monocytes, macrophages/microglia, and astrocytes in EAE. In addition, we show that EAE severity is increased in Lcn2−/− mice as compared with wild‐type controls. Finally, we demonstrate that elevated levels of Lcn2 are detected in the plasma and cerebrospinal fluid (CSF) in MS and in immune cells in CNS lesions in MS tissue sections. These data indicate that Lcn2 is a modulator of EAE pathogenesis and suggest that it may also play a role in MS.

Inflammatory Pathways in Spinal Cord Injury

Injury to the spinal cord results in direct damage to axons, neuronal cell bodies, and glia that cause functional loss below the site of injury. In addition, the injury also triggers an inflammatory response that contributes to secondary tissue damage that leads to further functional loss. Reducing inflammation after spinal cord injury (SCI) is therefore a worthy therapeutic goal. Inflammation in the injured spinal cord is a complex response that involves resident cells of the central nervous system as well as infiltrating immune cells, and is mediated by a variety of molecular pathways and signaling molecules. Here, we discuss approaches we have used to identify novel therapeutic targets to modulate the inflammatory response after SCI to reduce tissue damage and promote recovery. Effective treatments for SCI will likely require a combination of approaches to reduce inflammation and secondary damage with those that promote axon regeneration.

Arginase-1 is expressed exclusively by infiltrating myeloid cells in CNS injury and disease

Resident microglia and infiltrating myeloid cells play important roles in the onset, propagation, and resolution of inflammation in central nervous system (CNS) injury and disease. Identifying cell type-specific mechanisms will help to appropriately target interventions for tissue repair. Arginase-1 (Arg-1) is a well characterised modulator of tissue repair and its expression correlates with recovery after CNS injury. Here we assessed the cellular localisation of Arg-1 in two models of CNS damage. Using microglia specific antibodies, P2ry12 and Fc receptor-like S (FCRLS), we show the LysM-EGFP reporter mouse is an excellent model to distinguish infiltrating myeloid cells from resident microglia. We show that Arg-1 is expressed exclusively in infiltrating myeloid cells but not microglia in models of spinal cord injury (SCI) and experimental autoimmune encephalomyelitis (EAE). Our in vitro studies suggest that factors in the CNS environment prevent expression of Arg-1 in microglia in vivo. This work suggests different functional roles for these cells in CNS injury and repair and shows that such repair pathways can be switched on in infiltrating myeloid cells in pro-inflammatory environments.

Expression of iron homeostasis proteins in the spinal cord in experimental autoimmune encephalomyelitis and their implications for iron accumulation

Iron accumulation occurs in the CNS in multiple sclerosis (MS) and in experimental autoimmune encephalomyelitis (EAE). However, the mechanisms underlying such iron accumulation are not fully understood. We studied the expression and cellular localization of molecules involved in cellular iron influx, storage, and efflux. This was assessed in two mouse models of EAE: relapsing-remitting (RR-EAE) and chronic (CH-EAE). The expression of molecules involved in iron homeostasis was assessed at the onset, peak, remission/progressive and late stages of the disease. We provide several lines of evidence for iron accumulation in the EAE spinal cord which increases with disease progression and duration, is worse in CH-EAE, and is localized in macrophages and microglia. We also provide evidence that there is a disruption of the iron efflux mechanism in macrophages/microglia that underlie the iron accumulation seen in these cells. Macrophages/microglia also lack expression of the ferroxidases (ceruloplasmin and hephaestin) which have antioxidant effects. In contrast, astrocytes which do not accumulate iron, show robust expression of several iron influx and efflux proteins and the ferroxidase ceruloplasmin which detoxifies ferrous iron. Astrocytes therefore are capable of efficiently recycling iron from sites of EAE lesions likely into the circulation. We also provide evidence of marked dysregulation of mitochondrial function and energy metabolism genes, as well as of NADPH oxidase genes in the EAE spinal cord. This data provides the basis for the selective iron accumulation in macrophage/microglia and further evidence of severe mitochondrial dysfunction in EAE. It may provide insights into processes underling iron accumulation in MS and other neurodegenerative diseases in which iron accumulation occurs.

Microglia and macrophages differ in their inflammatory profile after permanent brain ischemia

ABSTRACT We studied the expression of pro‐ and anti‐inflammatory molecules in microglia and infiltrating monocyte‐derived macrophages after permanent Middle Cerebral Artery Occlusion (pMCAO). LysM‐EGFP knock‐in mice were used to distinguish between these two cell types, as peripheral myeloid cells are LysM‐EGFP+, while microglia are not. This was confirmed with P2ry12 (a microglial specific marker), Iba‐1 and EGFP immunostaining. The peak of LysM‐EGFP+ myeloid cell infiltration was 72 h post‐ischemia, and were distributed evenly in the lesion core, surrounded by a dense region of microglia. Flow cytometry showed that a higher percentage of microglia expressed TNF‐&agr; at 3 (24.3% vs 1.4%) and 7 (18.8% vs 3.4%) days post‐pMCAO as compared to infiltrating macrophages. Microglia and macrophages were purified by fluorescence activated cell sorting 72 h post‐ischemia to assess the mRNA expression of inflammatory markers. Macrophages upregulated expression of mRNA for arginase‐1 (Arg‐1) by 1000‐fold, and IL‐1&bgr; by 90‐fold as compared to microglia. At the protein level, a significantly number of macrophages expressed Arg‐1, while few if any microglia expressed Arg‐1. However, IL‐1&bgr; protein was not detected in macrophages by flow cytometry or immunofluorescence labeling of tissue sections. It was, however, detected in astrocytes along the lesion border. A PCR‐array screen of 84 inflammatory genes revealed that pro‐inflammatory chemokines and cytokines were predominantly upregulated in macrophages but down‐regulated in microglia in the ischemic brain. Our results show clear differences in the inflammatory expression profiles between microglia and macrophages 72 h post‐ischemia which may shape repair and pro‐regenerative mechanisms after stroke. HIGHLIGHTSLysM‐EGFP knock‐in mouse is a good tool to differentiate between microglia and infiltrating macrophages in the CNS.Microglia and macrophages after ischemic stroke differ in their inflammatory profile.Macrophages upregulate the expression of pro‐inflammatory and neutrophil recruitment genes.Microglia down‐regulate pro‐inflammatory and immune cell recruitment genes.Microglia in the peri‐infarct region, which express TNF‐&agr;, may help limit the expansion of the lesion.

Olfactory impairment and pathology in neurodegenerative disorders with brain iron accumulation.

Olfaction was tested using the 40-item smell identification test (UPSIT; for references see supplementary material online) in an appropriate cultural format for each patient (http://www.sensonics.com). Cognitive function was scored by mini-mental state examination. NBIA patients were enrolled if they were non-demented (MMSE > 24), able to speak and had no nasal pathology. UPSIT scores were compared between controls and patients with NBIA using a multivariate model adjusting for cognition. Post-mortem olfactory bulb tissue from a 56-year-old man with aceruloplasminemia was examined for iron (Perl’s stain; see supplementary material). Olfactory bulb, tract and cortex from 2 pathologically diagnosed NBIA cases (2 female, aged 77 and 65) and Neurodegenerative disorders with brain iron accumulation (NBIA) are a clinically and genetically heterogeneous group of neurodegenerative diseases featuring excessive brain iron deposition [2]. Iron deficient mice display impaired olfactory behaviour and chronic exposure to airborne iron particles is associated with hyposmia [4]. We hypothesised that dysregulated brain iron metabolism in NBIA might be associated with hyposmia. We tested olfactory function in a group of NBIA patients and performed histological examination of olfactory tissue from patients with NBIA and a mouse NBIA model for iron deposition.

Myeloid cell responses after spinal cord injury

The past decade has revealed much about the complexity of the local inflammatory response after spinal cord injury (SCI). A major challenge is to distinguish between microglia and monocyte-derived macrophages (MDMs) to determine their phenotype and function. Transcriptome studies have revealed microglia-selective genes but are still limited in scope because many markers are downregulated after injury. Additionally, new genetic reporter mice are available to study microglia and MDMs. There is more evidence now for the plasticity and heterogeneity of microglia and MDMs. We also discuss the role of neutrophils that are the first peripheral cells to enter the injured CNS.

SPARC and GluA1-Containing AMPA Receptors Promote Neuronal Health Following CNS Injury

The proper formation and maintenance of functional synapses in the central nervous system (CNS) requires communication between neurons and astrocytes and the ability of astrocytes to release neuromodulatory molecules. Previously, we described a novel role for the astrocyte-secreted matricellular protein SPARC (Secreted Protein, Acidic and Rich in Cysteine) in regulating α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) and plasticity at developing synapses. SPARC is highly expressed by astrocytes and microglia during CNS development but its level is reduced in adulthood. Interestingly, SPARC has been shown to be upregulated in CNS injury and disease. However, the role of SPARC upregulation in these contexts is not fully understood. In this study, we investigated the effect of chronic SPARC administration on glutamate receptors on mature hippocampal neuron cultures and following CNS injury. We found that SPARC treatment increased the number of GluA1-containing AMPARs at synapses and enhanced synaptic function. Furthermore, we determined that the increase in synaptic strength induced by SPARC could be inhibited by Philanthotoxin-433, a blocker of homomeric GluA1-containing AMPARs. We then investigated the effect of SPARC treatment on neuronal health in an injury context where SPARC expression is upregulated. We found that SPARC levels are increased in astrocytes and microglia following middle cerebral artery occlusion (MCAO) in vivo and oxygen-glucose deprivation (OGD) in vitro. Remarkably, chronic pre-treatment with SPARC prevented OGD-induced loss of synaptic GluA1. Furthermore, SPARC treatment reduced neuronal death through Philanthotoxin-433 sensitive GluA1 receptors. Taken together, this study suggests a novel role for SPARC and GluA1 in promoting neuronal health and recovery following CNS damage.

Peripherally derived macrophages modulate microglial function to reduce inflammation after CNS injury

Infiltrating monocyte-derived macrophages (MDMs) and resident microglia dominate central nervous system (CNS) injury sites. Differential roles for these cell populations after injury are beginning to be uncovered. Here, we show evidence that MDMs and microglia directly communicate with one another and differentially modulate each other’s functions. Importantly, microglia-mediated phagocytosis and inflammation are suppressed by infiltrating macrophages. In the context of spinal cord injury (SCI), preventing such communication increases microglial activation and worsens functional recovery. We suggest that macrophages entering the CNS provide a regulatory mechanism that controls acute and long-term microglia-mediated inflammation, which may drive damage in a variety of CNS conditions.