Anne C. Cotleur

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

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.

Selective Chemokine Receptor Usage by Central Nervous System Myeloid Cells in CCR2-Red Fluorescent Protein Knock-In Mice

Background Monocyte subpopulations distinguished by differential expression of chemokine receptors CCR2 and CX3CR1 are difficult to track in vivo, partly due to lack of CCR2 reagents. Methodology/Principal Findings We created CCR2-red fluorescent protein (RFP) knock-in mice and crossed them with CX3CR1-GFP mice to investigate monocyte subset trafficking. In mice with experimental autoimmune encephalomyelitis, CCR2 was critical for efficient intrathecal accumulation and localization of Ly6Chi/CCR2hi monocytes. Surprisingly, neutrophils, not Ly6Clo monocytes, largely replaced Ly6Chi cells in the central nervous system of these mice. CCR2-RFP expression allowed the first unequivocal distinction between infiltrating monocytes/macrophages from resident microglia. Conclusion/Significance These results refine the concept of monocyte subsets, provide mechanistic insight about monocyte entry into the central nervous system, and present a novel model for imaging and quantifying inflammatory myeloid populations.

TREM2 deficiency eliminates TREM2+ inflammatory macrophages and ameliorates pathology in Alzheimer’s disease mouse models

Jay and colleagues show that TREM2 deficiency reduces the number of macrophages infiltrating the brain and is protective against disease pathogenesis in mouse models of Alzheimer’s disease.

CXCR2-positive neutrophils are essential for cuprizone-induced demyelination: relevance to multiple sclerosis

Multiple sclerosis is an inflammatory demyelinating disorder of the CNS. Recent studies have suggested diverse mechanisms as underlying demyelination, including a subset of lesions induced by an interaction between metabolic insult to oligodendrocytes and inflammatory mediators. For mice of susceptible strains, cuprizone feeding results in oligodendrocyte cell loss and demyelination of the corpus callosum. Remyelination ensues and has been extensively studied. Cuprizone-induced demyelination remains incompletely characterized. We found that mice lacking the type 2 CXC chemokine receptor (CXCR2) were relatively resistant to cuprizone-induced demyelination and that circulating CXCR2-positive neutrophils were important for cuprizone-induced demyelination. Our findings support a two-hit process of cuprizone-induced demyelination, supporting the idea that multiple sclerosis pathogenesis features extensive oligodendrocyte cell loss. These data suggest that cuprizone-induced demyelination is useful for modeling certain aspects of multiple sclerosis pathogenesis.

Quantification of Self-Recognition in Multiple Sclerosis by Single-Cell Analysis of Cytokine Production

Identifying and quantifying autoaggressive responses in multiple sclerosis (MS) has been difficult in the past due to the low frequency of autoantigen-specific T cells, the high number of putative determinants on the autoantigens, and the different cytokine signatures of the autoreactive T cells. We used single-cell resolution enzyme-linked immunospot (ELISPOT) assays to study, directly ex vivo, proteolipid protein (PLP)-specific memory cell reactivity from MS patients and controls. Overlapping 9-aa-long peptides, spanning the entire PLP molecule in single amino acid steps, were used to determine the frequency and fine specificity of PLP-specific lymphocytes as measured by their IFN-γ and IL-5 production. MS patients (n = 22) responded to 4 times as many PLP peptides as did healthy controls (n = 22). The epitopes recognized in individual patients, up to 22 peptides, were scattered throughout the PLP molecule, showing considerable heterogeneity among MS patients. Frequency measurements showed that the number of PLP peptide-specific IFN-γ-producing cells averaged 11 times higher in MS patients than in controls. PLP peptide-induced IL-5-producing T cells occurred in very low frequencies in both MS patients and controls. This first comprehensive assessment of the anti-PLP-Th1/Th2 response in MS shows a greatly increased Th1 effector cell mass in MS patients. Moreover, the highly IFN-γ-polarized, IL-5-negative cytokine profile of the PLP-reactive T cells suggests that these cells are committed Th1 cells. The essential absence of uncommitted Th0 cells producing both cytokines may explain why therapeutic strategies that aim at the induction of immune deviation show little efficacy in the established disease.

Immunomodulatory effects of interferon beta-1a in multiple sclerosis

Several studies have established a role for interferon beta (IFNbeta) as a treatment for relapsing-remitting multiple sclerosis (MS). IFNbeta has been reported to decrease the relapse rate, relapse severity, progression of disability and development of new brain lesions. Its mechanisms of action, however, remain unclear. We hypothesize that immunomodulatory effects of IFNbeta may underlie its clinical efficacy. We used intracellular cytokine flow cytometry to analyze the effects of IFNbeta-1a on expression of the anti-inflammatory cytokine, IL-10, and its effects on major co-stimulatory molecules in MS patients. We found that peripheral blood mononuclear cells (PBMC) produced more IL-10 following in vitro or in vivo treatment with IFNbeta-1a. The primary cellular sources of IL-10 were monocytes and CD4(+) T lymphocytes. IL-10 production in response to IFNbeta-1a was increased in unseparated PBMC compared to purified lymphocyte cultures, indicating that interaction between monocytes and lymphocytes may influence IL-10 production in response to IFNbeta-1a. Using flow cytometry, we monitored the ex vivo expression of two major co-stimulatory pairs-B7/CD28 and CD40/CD40L-before and after intramuscular IFNbeta-1a treatment of MS patients. IFNbeta-1a lowered the expression of B7.1 on circulating B cells and increased B7.2 expression on monocytes. CD40 expression on B cells was down-regulated, but CD40 on monocytes was up-regulated by IFNbeta-1a treatment. These data suggest that co-stimulatory molecules are modulated by IFNbeta, providing a possible mechanism for its in vivo immune regulatory effects.

Sex differences in cytokine responses to myelin peptides in multiple sclerosis

Many autoimmune diseases preferentially affect women; however, the underlying mechanisms for the sex differences are poorly understood. We examined sex-dependent differences in the immunologic response to myelin proteins in 22 multiple sclerosis (MS) patients and 22 healthy controls. Using ELISA spot assay (ELISPOT) methodology, interferon (IFN) gamma and IL-5 secretions were examined at the single cell level in response to overlapping proteolipid protein (PLP) peptides. As previously reported, we observed an overall disease effect in the IFNgamma response, such that MS patients were significantly higher than controls. With respect to PLP-induced IFNgamma secretion, both MS and control females responded higher than their corresponding males. Female MS patients demonstrated the highest responses compared to MS males or healthy controls of either sex. Although MS females had high IFNgamma responses to PLP, they had no IL-5 responses at all, suggesting strong Th1 skewing. In contrast, MS males had more IL-5 than control males, who lacked IL-5 responses. These IL-5 responses suggested that disease and gender are not independent, but rather interact to influence the cytokine response to myelin. The data suggest a gender bias towards Th1 responses in MS, which may contribute to the female predominance in this disease.

Relationship between serum levels of IL-10, MRI activity and interferon beta-1a therapy in patients with relapsing remitting MS.

BACKGROUND The purposes of this study were to: (1) compare monthly serum IL-10 in patients with relapsing remitting (RR) multiple sclerosis (MS) and healthy controls, (2) determine the relationship between IL-10 and MRI activity and (3) determine the effect of interferon beta-1a (IFNB-1a) treatment on IL-10 levels. RESULTS Median serum IL-10 levels were lower in untreated RRMS (185.5 pg/ml) compared to controls (438.5 pg/ml) (P=0.19). Serum levels of IL-10 did not appear to predict the appearance of new gadolinium-enhancing (Gd+) lesions concurrently or 1 month thereafter. However, IL-10 levels were more likely to be elevated the month during which Gd+ lesions resolved (OR=3.14, P=0.01). Median IL-10 levels were lower during IFNB-1a treatment (P=0.01). CONCLUSIONS These observations suggest a relationship between serum IL-10 levels and resolution of abnormal vascular permeability in new lesions.

Interferon gamma responses to myelin peptides in multiple sclerosis correlate with a new clinical measure of disease progression

The relationship between autoreactivity to myelin antigens and disease progression in multiple sclerosis (MS) is not fully understood. We addressed this relationship by cross-sectionally comparing an objective measure of MS disability with immune cytokine responses to myelin proteins. The ELISPOT assay was used to determine the ex vivo interferon gamma (IFNgamma) and interleukin-10 (IL-10) production by peripheral blood mononuclear cells (PBMCs) in response to peptides spanning the entire proteolipid protein (PLP) and myelin basic protein (MBP) molecules in 20 patients with relapsing-remitting (RR) MS and 27 age- and sex-matched healthy controls. MS patients showed significantly higher MBP-induced IFNgamma responses and PLP-induced IL-10 responses compared with healthy controls. Using the Multiple Sclerosis Functional Composite (MSFC), a new multifactorial measure of disability, MS patients showed a significant correlation between the IFNgamma response to PLP peptides and MBP peptides, and disability. In contrast, in MS patients, there was no correlation between the MSFC and the response to unrelated control antigens or mitogens. These data show that myelin-specific T lymphocytes secreting the inflammatory cytokine IFNgamma correlate with functional impairment in MS, supporting an antigen-specific link between the immune response to myelin and disability in MS.

CXCL12-Induced Monocyte-Endothelial Interactions Promote Lymphocyte Transmigration Across an in Vitro Blood-Brain Barrier

An in vitro model of the human blood-brain barrier provides insights into how chemokine receptors regulate the transmigration of leukocytes into brain tissue. Going with the Flow White blood cells traffic ceaselessly throughout the body, using blood vessels as their conduits. They also migrate into inflamed tissues to defend the host against microbes or to repair damaged tissue. However, in many human diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis, or type 1 diabetes, white blood cells invade apparently healthy uninfected tissues and cause injury. Recent research has identified the molecular regulators (chemokine receptors and their chemokine ligands; adhesion molecules) of white blood cell transmigration out of the blood vessels and into tissues. Because there are 100 or so such molecules (used selectively when specific types of white blood cells transmigrate), it is important to identify those molecules that are most directly involved in harmful inflammation. Chemokine receptors are molecules on white blood cell surfaces that receive signals to guide cells into tissue, and they change as white blood cells transmigrate across different vessel walls. In a new study, Man and colleagues have devised an in vitro model of a specialized vessel wall of the human brain termed the blood-brain barrier (BBB). They use this elegant model to study how chemokine receptors influence and are influenced by the transmigration of white blood cells across a human BBB-like endothelial cell layer. First, the authors coaxed a special type of human endothelial cell to form a BBB-like layer in a dual perfusion chamber. Then, they allowed human white blood cells to flow across the layer at a flow rate approximating that found in brain capillaries. Some white blood cells flowed across the layer and out of the device, whereas others transmigrated across the BBB-like endothelial layer into the lower chamber of the device. The researchers wanted to establish how chemokine receptor expression by white blood cells would alter as the cells transmigrated across the BBB. They studied a chemokine receptor termed CXCR4, which is expressed on almost all white blood cells. When they added the triggering molecule for CXCR4 to their in vitro system, unexpectedly, they found that only one cell type, monocytes, showed altered CXCR4 expression. CXCR4 appeared to deliver signals to monocytes, which empowered these cells to assist other white blood cells such as T and B cells to cross the BBB. This surprising result opens up new vistas for understanding how white blood cells and vessel wall endothelial cells “talk” to each other in inflamed tissues and should spur progress for identifying the best targets for blocking harmful inflammation in the brain. The accumulation of inflammatory cells in the brain parenchyma is a critical step in the pathogenesis of neuroinflammatory diseases such as multiple sclerosis (MS). Chemokines and adhesion molecules orchestrate leukocyte transmigration across the blood-brain barrier (BBB), but the dynamics of chemokine receptor expression during leukocyte transmigration are unclear. We describe an in vitro BBB model system using human brain microvascular endothelial cells that incorporates shear forces mimicking blood flow to elucidate how chemokine receptor expression is modulated during leukocyte transmigration. In the presence of the chemokine CXCL12, we examined modulation of its receptor CXCR4 on human T cells, B cells, and monocytes transmigrating across the BBB under flow conditions. CXCL12 stimulated transmigration of CD4+ and CD8+ T cells, CD19+ B cells, and CD14+ monocytes. Transmigration was blocked by CXCR4-neutralizing antibodies. Unexpectedly, CXCL12 selectively down-regulated CXCR4 on transmigrating monocytes, but not T cells. Monocytes underwent preferential CXCL12-mediated adhesion to the BBB in vitro compared with lymphocytes. These findings provide new insights into leukocyte-endothelial interactions at the BBB under conditions mimicking blood flow and suggest that in vitro BBB models may be useful for identifying chemokine receptors that could be modulated therapeutically to reduce neuroinflammation in diseases such as MS.