Macrophage galactose-type lectin (MGL) is induced on M2 microglia and participates in the resolution phase of autoimmune neuroinflammation.

BACKGROUND: Multiple sclerosis (MS) involves a misdirected immune attack against myelin in the brain and spinal cord, leading to profound neuroinflammation and neurodegeneration. While the mechanisms of disease pathogenesis have been widely studied, the suppression mechanisms that lead to the resolution of the autoimmune response are still poorly understood. Here, we investigated the role of the C-type lectin receptor macrophage galactose-type lectin (MGL), usually expressed on tolerogenic antigen-presenting cells (APCs), as a negative regulator of autoimmune-driven neuroinflammation. METHODS: We used in silico, immunohistochemical, immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR) and flow cytometry analysis to explore the expression and functionality of MGL in human macrophages and microglia, as well as in MS post-mortem tissue. In vitro, we studied the capacity of MGL to mediate apoptosis of experimental autoimmune encephalomyelitis (EAE)-derived T cells and mouse CD4+ T cells. Finally, we evaluated in vivo and ex vivo the immunomodulatory potential of MGL in EAE. RESULTS: MGL plays a critical role in the resolution phase of EAE as MGL1-deficient (Clec10a-/-) mice showed a similar day of onset but experienced a higher clinical score to that of WT littermates. We demonstrate that the mouse ortholog MGL1 induces apoptosis of autoreactive T cells and diminishes the expression of pro-inflammatory cytokines and inflammatory autoantibodies. Moreover, we show that MGL1 but not MGL2 induces apoptosis of activated mouse CD4+ T cells in vitro. In human settings, we show that MGL expression is increased in active MS lesions and on alternatively activated microglia and macrophages which, in turn, induces the secretion of the immunoregulatory cytokine IL-10, underscoring the clinical relevance of this lectin. CONCLUSIONS: Our results show a new role of MGL-expressing APCs as an anti-inflammatory mechanism in autoimmune neuroinflammation by dampening pathogenic T and B cell responses, uncovering a novel clue for neuroprotective therapeutic strategies with relevance for in MS clinical applications.

Human Milk Blocks DC-SIGN-Pathogen Interaction via MUC1.

Beneficial effects of breastfeeding are well-recognized and include both immediate neonatal protection against pathogens and long-term protection against allergies and autoimmune diseases. Although several proteins have been identified to have anti-viral or anti-bacterial effects like secretory IgA or lactoferrin, the mechanisms of immune modulation are not fully understood. Recent studies identified important beneficial effects of glycans in human milk, such as those expressed in oligosaccharides or on glycoproteins. Glycans are recognized by the carbohydrate receptors C-type lectins on dendritic cell (DC) and specific tissue macrophages, which exert important functions in immune modulation and immune homeostasis. A well-characterized C-type lectin is dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), which binds terminal fucose. The present study shows that in human milk, MUC1 is the major milk glycoprotein that binds to the lectin domain of DC-SIGN and prevents pathogen interaction through the presence of Lewis x-type oligosaccharides. Surprisingly, this was specific for human milk, as formula, bovine or camel milk did not show any presence of proteins that interacted with DC-SIGN. The expression of DC-SIGN is found in young infants along the entire gastrointestinal tract. Our data thus suggest the importance of human milk glycoproteins for blocking pathogen interaction to DC in young children. Moreover, a potential benefit of human milk later in life in shaping the infants immune system through DC-SIGN cannot be ruled out.

Disturbed function of the blood-cerebrospinal fluid barrier aggravates neuro-inflammation.

Multiple sclerosis (MS) is a chronic neuro-inflammatory disorder, which is marked by the invasion of the central nervous system by monocyte-derived macrophages and autoreactive T cells across the brain vasculature. Data from experimental animal models recently implied that the passage of leukocytes across the brain vasculature is preceded by their traversal across the blood-cerebrospinal fluid barrier (BCSFB) of the choroid plexus. The correlation between the presence of leukocytes in the CSF of patients suffering from MS and the number of inflammatory lesions as detected by magnetic resonance imaging suggests that inflammation at the choroid plexus contributes to the disease, although in a yet unknown fashion. We here provide first insights into the involvement of the choroid plexus in the onset and severity of the disease and in particular address the role of the tight junction protein claudin-3 (CLDN3) in this process. Detailed analysis of human post-mortem brain tissue revealed a selective loss of CLDN3 at the choroid plexus in MS patients compared to control tissues. Importantly, mice that lack CLDN3 have an impaired BCSFB and experience a more rapid onset and exacerbated clinical signs of experimental autoimmune encephalomyelitis, which coincides with enhanced levels of infiltrated leukocytes in their CSF. Together, this study highlights a profound role for the choroid plexus in the pathogenesis of multiple sclerosis, and implies that CLDN3 may be regarded as a crucial and novel determinant of BCSFB integrity.

Selective upregulation of scavenger receptors in and around demyelinating areas in multiple sclerosis.

Autoantibodies and complement opsonization have been implicated in the process of demyelination in the major human CNS demyelinating disease multiple sclerosis (MS), but scavenger receptors (SRs) may also play pathogenetic roles. We characterized SR mRNA and protein expression in postmortem brain tissue from 13 MS patients in relation to active demyelination. CD68, chemokine (C-X-C motif) ligand 16 (CXCL16), class A macrophage SR (SR-AI/II), LOX-1 (lectin-like oxidized low-density lipoprotein receptor 1), FcγRIII, and LRP-1 (low-density lipoprotein receptor-related protein 1) mRNA were upregulated in the rims of chronic active MS lesions. CD68 and CXCL16 mRNA were also upregulated around chronic active MS lesions. By immunohistochemistry, CD68, CXCL16, and SR-AI/II were expressed by foamy macrophages in the rim and by ramified microglia around chronic active MS lesions. CXCL16 and SR-AI/II were also expressed by astrocytes in MS lesions and by primary human microglia and astrocytes in vitro. These data suggest that SRs are involved in myelin uptake in MS, and that upregulation of CD68, CXCL16, and SR-AI/II is one of the initial events in microglia as they initiate myelin phagocytosis. As demyelination continues, additional upregulation of LOX-1, FcγRIII, and LRP-1 may facilitate this process.

Glycodendrimers prevent HIV transmission via DC-SIGN on dendritic cells.

Dendritic cells (DCs) are antigen-presenting cells efficient in capturing pathogens, and processing their antigenic determinants for presentation to antigen-specific T cells to induce robust immune responses. Their location at peripheral tissues and the expression of pattern-recognition receptors, among them DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), facilitates the capture of pathogens before spreading. However, some pathogens have developed strategies to escape the immune system. One of the most successful is HIV-1, which targets DC-SIGN for transport to the lymph node where the virus infects CD4(+) T cells. Contact of HIV-1 with DC-SIGN is thus the first event in the pathogenic cascade and, therefore, it is the primary target point for therapies aimed at HIV infection prevention. DC-SIGN recognizes specific glycans on HIV-1 and this interaction can be blocked by competitive inhibition through glycans. Although the affinity of glycans is relatively low, multivalency may increase avidity and the strength to compete with HIV-1 virions. We have designed multivalent dendrimeric compounds based on Lewis-type antigens that bind DC-SIGN with high selectivity and avidity and that effectively block gp120 binding to DC-SIGN and, consequently, HIV transmission to CD4(+) T cells. Binding to DC-SIGN and gp120 inhibition was higher on glycodendrimers with larger molecular diameter, indicating that the geometry of the compounds is an important factor determining their functionality. Our compounds elicited DC-SIGN internalization, a property of the receptor upon triggering, but did not affect the maturation status of DCs. Thus, Le(X) glycodendrimers could be incorporated into topic prophylactic approaches for the prevention of HIV-1 transmission.

Phenotyping primary human microglia: tight regulation of LPS responsiveness.

Much is still unknown about mechanisms underlying the phenotypical and functional versatility of human microglia. Therefore, we developed a rapid procedure to isolate pure microglia from postmortem human brain tissue and studied their immediate ex vivo phenotype and responses to key inflammatory mediators. Microglia were isolated, along with macrophages from the choroid plexus by tissue dissociation, density gradient separation, and selection with magnetic microbeads. By flow cytometry, microglia were identified by a CD11b(+) CD45(dim) phenotype and a smaller size compared with CD11b(+) CD45(high) macrophages. Interestingly, white matter microglia from donors with peripheral inflammation displayed elevated CD45 levels and increased size and granularity, but were still distinct from macrophages. The phenotype of isolated microglia was further specified by absent surface expression of CD14, CD200 receptor, and mannose receptor (MR, CD206), all of which were markedly expressed by macrophages. Microglia stimulated immediately after isolation with LPS and IFNγ failed to upregulate TNFα or CCR7. Notably, responsiveness to LPS and IFNγ was clearly instigated in microglia after overnight preculture, which coincided with a strong upregulation of CD14. Culture of microglia with IL-4 resulted in the induction of HLA-DR and CCL18 but not MR, whereas culture with dexamethasone did induce MR, in addition to CD163 and CCL18. In conclusion, this study demonstrates phenotypic changes of microglia associated with peripheral inflammation, and reveals tight regulation of responses to LPS and IFNγ as well as distinct microglial responses to IL-4 and glucocorticoids. These findings are of high relevance to studies on human microglia functioning in health and disease.

Expression of the inhibitory CD200 receptor is associated with alternative macrophage activation.

Classical macrophage activation is inhibited by the CD200 receptor (CD200R). Here, we show that CD200R expression was specifically induced on human in vitro polarized macrophages of the alternatively activated M2a subtype, generated by incubation with IL-4 or IL-13. In mice, peritoneal M2 macrophages, elicited during infection with the parasites Taenia crassiceps or Trypanosoma brucei brucei, expressed increased CD200R levels compared to those derived from uninfected mice. However, in vitrostimulation of mouse peritoneal macrophages and T. crassiceps infection in IL-4-/- and IL-4R-/- mice showed that, in contrast to humans, induction of CD200R in mice was not IL-4 or IL-13 dependent. Our data identify CD200R as a suitable marker for alternatively activated macrophages in humans and corroborate observations of distinct species- and/or site-specific mechanisms regulating macrophage polarization in mouse and man.

Restoring immune suppression in the multiple sclerosis brain.

Multiple sclerosis is a very disabling inflammatory demyelinating disease of the brain of unknown etiology. Current therapies can reduce new lesion development and partially prevent clinical disease activity, but none can halt the progression, or cure the disease. We will review current therapeutic strategies, which are mostly discussed in literature in terms of their effective inhibition of T cells. However, we argue that many of these treatments also influence the myeloid compartment. Interestingly, recent evidence indicates that myelin phagocytosis by infiltrated macrophages and activated microglia is not just a hallmark of multiple sclerosis, but also a key determinant of lesion development and disease progression. We reason that severe side effects and/or insufficient effectiveness of current treatments necessitates the search for novel therapeutic targets, and postulate that these should aim at manipulation of the activation and phagocytic capacity of macrophages and microglia. We will discuss three candidate targets with high potential, namely the complement receptor 3, CD47-SIRPalpha interaction as well as CD200-CD200R interaction. Blocking the actions of complement receptor 3 could inhibit myelin phagocytosis, as well as migration of myeloid cells into the brain. CD47 and CD200 are known to inhibit macrophage/microglia activation through binding to their receptors SIRPalpha and CD200R, expressed on phagocytes. Triggering these receptors may thus dampen the inflammatory response. Our recent findings indicate that the CD200-CD200R interaction is the most specific and hence probably best-suited target to suppress excessive macrophage and microglia activation, and restore immune suppression in the brain of patients with multiple sclerosis.

GITR triggering induces expansion of both effector and regulatory CD4+ T cells in vivo.

Glucocorticoid-induced TNF receptor family-related protein (GITR) is expressed on activated and regulatory T cells, but its role on these functionally opposing cell types is not fully understood. Here we describe that transgenic expression of GITR's unique ligand (GITRL) induces a prominent increase of both effector and regulatory CD4(+) T cells, but not CD8(+) T cells. Regulatory T cells from GITRL transgenic mice are phenotypically activated and retain their suppressive capacity. The accumulation of effector and regulatory T cells is not due to enhanced differentiation of naive T cells, but is a direct result of increased proliferation. Functional consequences of increased numbers of both regulatory and effector T cells were tested in an autoimmune model and show that GITR stimulation is protective, as it significantly delays disease induction. These data indicate that GITR regulates the balance between regulatory and effector CD4(+) T cells by enhancing proliferation of both populations in parallel.

Distribution of the immune inhibitory molecules CD200 and CD200R in the normal central nervous system and multiple sclerosis lesions suggests neuron-glia and glia-glia interactions.

CD200 is a membrane glycoprotein that suppresses immune activity via its receptor, CD200R. CD200-CD200R interactions have recently been considered to contribute to the "immune privileged" status of the central nervous system (CNS). The mechanisms by which these interactions take place are not well understood in part because there is limited detailed information on the distribution of CD200 and CD200R in the CNS. Here, we used immunohistochemistry to characterize the distinct anatomical and cellular distribution of these molecules in multiple sclerosis (MS) lesions and controls. CD200 was robustly expressed in gray matter areas including the cerebral cortex, hippocampus, striatum, cerebellum, and spinal cord, where neurons appeared immunopositive. CD200 expression was also detected in oligodendrocytes, but not in astrocytes or microglia. In CNS samples from MS patients, CD200 expression was additionally observed on reactive astrocytes in chronic active plaque centers, despite our previous finding of an overall decrease ofCD200 expression in MS lesions. In contrast to CD200, the immunolocalization pattern of CD200R was very distinct, showing high expression on perivascular macrophages in both gray and white matter. Using flow cytometry, we also found that human primary microglia express low levels of CD200R. These data suggest that CD200-mediated immune suppression may occur not only via neuron-microglia interactions, but also via glia-glia interactions, especially in inflammatory conditions in which an immune-suppressive environment needs to be restored; this may occur as a result of increased CD200 expression on reactive astrocytes.

Downregulation of macrophage inhibitory molecules in multiple sclerosis lesions.

OBJECTIVE: Inflammatory and demyelinating activity of activated resident macrophages (microglia) and recruited blood-borne macrophages are considered crucial in multiple sclerosis (MS) lesion development. The membrane glycoproteins CD200 and CD47, highly expressed on neurons, are mediators of macrophage inhibition via their receptors CD200R and signal-regulatory protein alpha, respectively, on myeloid cells. We determined the expression pattern of immune inhibitory molecules in relation to genes involved in macrophage activation and MS lesion pathology. METHODS: Laser dissection microscopy was combined with real-time polymerase chain reaction to quantitatively study these gene expression patterns in specific subareas (rim, center, and normal-appearing white matter) of chronic active and inactive MS lesions. RESULTS: Hallmarks of MS pathology were confirmed by messenger RNA expression patterns of glial fibrillary acidic protein, neurofilament (NF), myelin basic protein, growth factors, chemokines and receptors, and macrophage activation markers, although expression of osteopontin and alphaB-crystallin was decreased. CD200 and CD47 were downregulated in the center of chronic active and inactive MS lesions. CD47 expression was also decreased in the rim of chronic active lesions, where complement expression was increased. This expression profile was also found in normal-appearing white matter surrounding these lesions, but not surrounding inactive lesions. Expression of CD200R and signal-regulatory protein alpha was not altered. INTERPRETATION: These data suggest that diminished immune inhibition via decreased CD200 and CD47 expression contributes to a disturbed equilibrium in macrophage and microglia activation in MS lesions. Furthermore, this may result in a proinflammatory predisposition in the area surrounding chronic active lesions, thereby contributing to axonal injury, demyelination, and possible lesion expansion.

EMR1, the human homolog of F4/80, is an eosinophil-specific receptor.

The EGF-TM7 F4/80 is a defining marker of murine macrophage populations. Applying flow cytometric analysis using the newly generated mAb A10, and quantitative real-time PCR, we here report the surprising observation that the human ortholog of F4/80, EGF-like module containing mucin-like hormone receptor (EMR)1, is absent on mononuclear phagocytic cells including monocytes, macrophages, and myeloid dendritic cells. Unexpectedly, we found that EMR1 expression is restricted to eosinophilic granulocytes, where expression is overlapping with the eotaxin receptor CCR3 and the immunoglobulin-like lectin Siglec-8. Absence on other leukocytes, including basophils, implies that EMR1 is a highly specific marker for eosinophils in humans.