Agnieszka Wlodarczyk

Comparison of microglia and infiltrating CD11c + cells as antigen presenting cells for T cell proliferation and cytokine response

BackgroundTissue-resident antigen-presenting cells (APC) exert a major influence on the local immune environment. Microglia are resident myeloid cells in the central nervous system (CNS), deriving from early post-embryonic precursors, distinct from adult hematopoietic lineages. Dendritic cells (DC) and macrophages infiltrate the CNS during experimental autoimmune encephalomyelitis (EAE). Microglia are not considered to be as effective APC as DC or macrophages.MethodsIn this work we compared the antigen presenting capacity of CD11c+ and CD11c− microglia subsets with infiltrating CD11c+ APC, which include DC. The microglial subpopulations (CD11c− CD45dim CD11b+ and CD11c+ CD45dim CD11b+) as well as infiltrating CD11c+ CD45high cells were sorted from CNS of C57BL/6 mice with EAE. Sorted cells were characterised by flow cytometry for surface phenotype and by quantitative real-time PCR for cytokine expression. They were co-cultured with primed T cells to measure induction of T cell proliferation and cytokine response.ResultsThe number of CD11c+ microglia cells increased dramatically in EAE. They expressed equivalent levels of major histocompatibility complex and co-stimulatory ligands CD80 and CD86 as those expressed by CD11c+ cells infiltrating from blood. CD11c+ microglia differed significantly from blood-derived CD11c+ cells in their cytokine profile, expressing no detectable IL-6, IL-12 or IL-23, and low levels of IL-1β. By contrast, CD11c− microglia expressed low but detectable levels of all these cytokines. Transforming growth factor β expression was similar in all three populations. Although CNS-resident and blood-derived CD11c+ cells showed equivalent ability to induce proliferation of myelin oligodendrocyte glycoprotein-immunised CD4+ T cells, CD11c+ microglia induced lower levels of T helper (Th)1 and Th17 cytokines, and did not induce Th2 cytokines.ConclusionsOur findings show distinct subtypes of APC in the inflamed CNS, with a hierarchy of functional competence for induction of CD4+ T cell responses.

Interferons in the central nervous system: A few instruments play many tunes

Interferons (IFNs) are implicated as an important component of the innate immune system influencing viral infections, inflammation, and immune surveillance. We review here the complex biological activity of IFNs in the central nervous system (CNS) and associated glial–immune interactions, with focus specifically on the Type I IFNs in physiological and pathological conditions. IFN‐α and IFN‐β are the predominant Type I IFNs in the CNS. They are produced in the CNS by glial cells, mostly microglia and astrocytes, as well as by neurons. A variety of mechanisms stimulate IFN production in glial cells, including innate stimuli from Toll‐like and other receptors, which can recognize endogenous entities, as well as pathogens. We will review evidence that differential signaling by IFN‐α versus IFN‐β through the common heterodimeric receptor IFNAR is the basis for CNS‐selective Type I IFN response, and the capacity of CNS glial cells to produce and respond to Type I IFN. Differential signaling outcomes of IFN‐α and IFN‐β, which have been ascribed to differential affinity for IFNAR1 and IFNAR2, determine whether Type I IFN exert pathogenic or protective roles in the CNS. These points will be discussed with reference to selected neurological diseases, and effects of Type I IFN on the integrity of the blood–brain barrier. GLIA 2014;62:339–355

A novel microglial subset plays a key role in myelinogenesis in developing brain

Microglia are resident macrophages of the central nervous system that contribute to homeostasis and neuroinflammation. Although known to play an important role in brain development, their exact function has not been fully described. Here, we show that in contrast to healthy adult and inflammation‐activated cells, neonatal microglia show a unique myelinogenic and neurogenic phenotype. A CD11c+ microglial subset that predominates in primary myelinating areas of the developing brain expresses genes for neuronal and glial survival, migration, and differentiation. These cells are the major source of insulin‐like growth factor 1, and its selective depletion from CD11c+ microglia leads to impairment of primary myelination. CD11c‐targeted toxin regimens induced a selective transcriptional response in neonates, distinct from adult microglia. CD11c+ microglia are also found in clusters of repopulating microglia after experimental ablation and in neuroinflammation in adult mice, but despite some similarities, they do not recapitulate neonatal microglial characteristics. We therefore identify a unique phenotype of neonatal microglia that deliver signals necessary for myelination and neurogenesis.

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

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.

Thymic CCL2 influences induction of T-cell tolerance

Thymic epithelial cells (TEC) and dendritic cells (DC) play a role in T cell development by controlling the selection of the T cell receptor repertoire. DC have been described to take up antigens in the periphery and migrate into the thymus where they mediate tolerance via deletion of autoreactive T cells, or by induction of natural regulatory T cells. Migration of DC to thymus is driven by chemokine receptors. CCL2, a major ligand for the chemokine receptor CCR2, is an inflammation-associated chemokine that induces the recruitment of immune cells in tissues. CCL2 and CCR2 are implicated in promoting experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis. We here show that CCL2 is constitutively expressed by endothelial cells and TEC in the thymus. Transgenic mice overexpressing CCL2 in the thymus showed an increased number of thymic plasmacytoid DC and pronounced impairment of T cell development. Consequently, CCL2 transgenic mice were resistant to EAE. These findings demonstrate that expression of CCL2 in thymus regulates DC homeostasis and controls development of autoreactive T cells, thus preventing development of autoimmune diseases.

Neuromyelitis optica-like pathology is dependent on type I interferon response.

Neuromyelitis optica is an antibody-mediated autoimmune inflammatory disease of the central nervous system. Reports have suggested that interferon beta which is beneficial for multiple sclerosis, exacerbates neuromyelitis optica. Our aim was to determine whether type I interferon plays a role in the formation of neuromyelitis optica lesions. Immunoglobulin G from a neuromyelitis optica patient was injected intracerebrally with human complement to type I interferon receptor deficient and wildtype mice. Loss of aquaporin-4 and glial fibrillary acidic protein was reduced in type I interferon receptor deficient mice brain. Our findings suggest that type I interferon signaling contributes to neuromyelitis optica pathogenesis.

The chemokine receptor CCR2 maintains plasmacytoid dendritic cell homeostasis

Thymic dendritic cells (DC) play a role in central tolerance. Three thymic DC subtypes have been described: plasmacytoid DC (pDC) and two conventional DC (cDC), CD8α+ Sirpα- DC and Sirpα+ CD8α- cDC. Both pDC and Sirpα+ cDC can take up antigen in periphery and migrate into the thymus in response to chemokine signaling via CCR9 and CCR2 respectively. CCL2 is a major ligand for CCR2 and we previously showed that it was constitutively expressed in thymus, and that mice overexpressing CCL2 in thymus had reduced numbers of autoreactive T cells but elevated numbers of pDC. We have here investigated the role of CCL2-CCR2 axis in thymic pDC migration. We found that pDC expressed CCR2 at a high level and that their frequency was decreased in thymus, spleen and inguinal lymph nodes in mice lacking CCR2, but not in mice lacking CCL2. pDC migration towards the cortex or medulla within the thymus was not affected by CCL2 or CCR2 deficiency. Although some thymic progenitors expressed CCR2, this did not include those that give rise to pDC. Based on these results, we propose that CCR2 is involved in pDC homeostasis but its ligand CCL2 does not play a major role.

Experimental demyelination and axonal loss are reduced in MicroRNA-146a deficient mice

Background The cuprizone (CPZ) model of multiple sclerosis (MS) was used to identify microRNAs (miRNAs) related to in vivo de- and remyelination. We further investigated the role of miR-146a in miR-146a-deficient (KO) mice: this miRNA is differentially expressed in MS lesions and promotes differentiation of oligodendrocyte precursor cells (OPCs) during remyelination, but its role has not been examined during demyelination. Methods MicroRNAs were examined by Agilent Mouse miRNA Microarray in the corpus callosum during CPZ-induced demyelination and remyelination. Demyelination, axonal loss, changes in number of oligodendrocytes, OPCs, and macrophages/microglia was compared by histology/immunohistochemistry between KO and WT mice. Differential expression of target genes and proteins of miR-146a was analyzed in the transcriptome (4 × 44K Agilent Whole Mouse Genome Microarray) and proteome (liquid chromatography tandem mass spectrometry) of CPZ-induced de- and remyelination in WT mice. Levels of proinflammatory molecules in the corpus callosum were compared in WT versus KO mice by Meso Scale Discovery multiplex protein analysis. Results miR-146a was increasingly upregulated during CPZ-induced de- and remyelination. The absence of miR-146a in KO mice protected against demyelination, axonal loss, body weight loss, and atrophy of thymus and spleen. The number of CNP+ oligodendrocytes was increased during demyelination in the miR-146a KO mice, while there was a trend of increased number of NG2+ OPCs in the WT mice. miR-146a target genes, SNAP25 and SMAD4, were downregulated in the proteome of demyelinating corpus callosum in WT mice. Higher levels of SNAP25 were measured by ELISA in the corpus callosum of miR-146a KO mice, but there was no difference between KO and WT mice during demyelination. Multiplex protein analysis of the corpus callosum lysate revealed upregulated TNF-RI, TNF-RII, and CCL2 in the WT mice in contrast to KO mice. The number of Mac3+ and Iba1+ macrophages/microglia was reduced in the demyelinating corpus callosum of the KO mice. Conclusion During demyelination, absence of miR-146a reduced inflammatory responses, demyelination, axonal loss, the number of infiltrating macrophages, and increased the number of myelinating oligodendrocytes. The number of OPCs was slightly higher in the WT mice during remyelination, indicating a complex role of miR-146a during in vivo de- and remyelination.

CCL2 recruits T cells into the brain in a CCR2-independent manner

CCL2 is a chemokine that can be induced during neuroinflammation to recruit immune cells, but its role in the central nervous system (CNS) is unclear. Our aim was to better understand its role. We induced CCL2 in CNS of naive CCL2‐deficient mice using intrathecally administered replication‐defective adenovirus and examined cell infiltration by flow cytometry. CCL2 expression induced pronounced and unexpected recruitment of regulatory and IFNγ‐producing T cells to CNS from blood, possibly related to defective egress of monocytes from CCL2‐deficient bone marrow. Infiltration also occurred in mice lacking CCR2, a receptor for CCL2. Expression of another receptor for CCL2, CCR4, and CXCR3, a receptor for CXCL10, which was also induced, were both increased in CCL2‐treated CNS. CCR4 was expressed by neurons and astrocytes as well as CD4 T cells, and CXCR3 was expressed by CD4 and CD8 T cells. Chemokine‐recruited T cells did not lead to CNS pathology. Our findings show a role for CCL2 in recruitment of CD4 T cells to the CNS and show that redundancy among chemokine receptors ensures optimal response.