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.

Moss-based production of asialo-erythropoietin devoid of Lewis A and other plant-typical carbohydrate determinants.

Protein therapeutics represent one of the most increasing areas in the pharmaceutical industry. Plants gain acceptance as attractive alternatives for high-quality and economical protein production. However, as the majority of biopharmaceuticals are glycoproteins, plant-specific N-glycosylation has to be taken into consideration. In Physcomitrella patens (moss), glyco-engineering is an applicable tool, and the removal of immunogenic core xylose and fucose residues was realized before. Here, we present the identification of the enzymes that are responsible for terminal glycosylation (α1,4 fucosylation and ß1,3 galactosylation) on complex-type N-glycans in moss. The terminal trisaccharide consisting of α1,4 fucose and ß1,3 galactose linked to N-acetylglucosamine forms the so-called Lewis A epitope. This epitope is rare on moss wild-type proteins, but was shown to be enriched on complex-type N-glycans of moss-produced recombinant human erythropoietin, while unknown from the native human protein. Via gene targeting of moss galactosyltransferase and fucosyltransferase genes, we identified the gene responsible for terminal glycosylation and were able to completely abolish the formation of Lewis A residues on the recombinant biopharmaceutical.

N-Glycosylation engineering of tobacco plants to produce asialoerythropoietin.

UNLABELLED: Erythropoietin (EPO) is a glycoprotein hormone that displays both hematopoietic and tissue-protective functions by binding to two distinct receptors. Recombinant human EPO (rhuEPO) is widely used for the treatment of anemia, but its use for tissue protection is limited because of potentially harmful increases in red blood cell mass when higher doses of rhuEPO are used. Recent studies have shown that asialoerythropoietin (asialo-rhuEPO), a desialylated form of rhuEPO, lacks hematopoietic activity, but retains cytoprotective activity. Currently, a small amount of asialo-rhuEPO is produced by enzymatic desialylation of rhuEPO. The prohibitive cost of rhuEPO, however, is a major limitation of this method. Plants have the ability to synthesize complex N-glycans, but lack enzymatic activities to add sialic acid and ß1,4-galactose to N-glycan chains. Plants could be genetically engineered to produce asialo-rhuEPO by introducing human ß1,4-galactosyltransferase. The penultimate ß1,4-linked galactose residues are important for in vivo biological activity. In this proof of concept study, we show that tobacco plants co-expressing human ß1,4-galactosyltransferase and EPO genes accumulated asialo-rhuEPO. Purified asialo-rhuEPO binds to an Erythrina cristagalli lectin column, indicating that its N-glycan chains bear terminal ß1,4-galactose residues and that the co-expressed GalT is functionally active. Asialo-rhuEPO interacted with the EPO receptor (EPOR) with similar affinity as rhuEPO, implying that it was properly folded. The strategy described here provides a straightforward way to produce asialo-rhuEPO for research and therapeutic purposes. KEY MESSAGE: N-glycosylation pathway in tobacco plants could be genetically engineered to produce a tissue-protective cytokine, asialoerythropoietin (a desialylated form of human hormone erythropoietin).

The dermal microenvironment induces the expression of the alternative activation marker CD301/mMGL in mononuclear phagocytes, independent of IL-4/IL-13 signaling.

Recently, we have shown that mononuclear phagocytes comprise the majority of interstitial cells in the mouse dermis, as indicated by their phenotypic and functional characteristics. In particular, these cells express the mouse macrophage galactose-/N-acetylgalactosamine-specific-lectin (mMGL)/CD301, identified by the monoclonal antibody ER-MP23, as well as other macrophage markers. As expression of mMGL is induced by IL-4 or IL-13 and is therefore a marker of alternatively activated macrophages, we asked whether dermal mononuclear phagocytes are genuinely alternatively activated. We observed that these cells expressed, next to mMGL, two other alternative activation markers, namely, the mannose receptor/CD206 and Dectin-1. Yet, as this expression profile was similar in IL-4 receptor alpha knockout mice, neither IL-4 nor IL-13 signaling appeared to be required for this phenotype. We also found that Langerhans cells (LC), which showed only a low level of mMGL in the epidermis, up-regulated mMGL expression upon migration through the dermis, allowing these cells to internalize limited amounts of mMGL ligands. LC isolated from epidermal preparations did not show this up-regulation when cultured in standard medium, but whole skin-conditioned medium did stimulate mMGL expression by LC. The vast majority of mMGL molecules was present in the cytoplasm, however. LC, which arrived in skin-draining lymph nodes, quickly down-regulated mMGL expression, and dermally derived cells retained significant mMGL levels. Taken together, these data suggest that the dermal microenvironment induces mononuclear phagocyte subpopulations to express mMGL and possibly other markers of alternatively activated macrophages, independent of IL-4/IL-13 signaling.

Retinoic acid modulates the asialoglycoprotein receptor expression in cultured fetal rat hepatocytes.

The influence of retinoic acid on the expression of a typical marker of hepatocyte differentiation, i.e. the asialoglycoprotein receptor, has been studied. Cultured hepatocytes, isolated from adult rats, a model of quiescent, mature cells and from 20-day-old fetuses, a model of proliferating and less differentiated cells, were used. The asialoglycoprotein receptor expression appears to be affected by retinoic acid during prenatal life; both mRNA level and protein amount increased in fetal hepatocytes, but no modification has been found in adult cells, suggesting a regulative effect of retinoic acid during prenatal life, acting at transcriptional and/or translational level. Surprisingly, the receptor binding activity of adult hepatocytes is decreased after retinoic acid treatment, indicating a possible further modulation by this molecule on receptor activity at the post-translational level.

Incorporation of sialic acid into Trypanosoma cruzi macromolecules. A proposal for a new metabolic route.

Sialo- and asialoglycoconjugates were isolated from Trypanosoma cruzi epimastigotes and their composition determined. Sialoglycoconjugates bound to wheat germ agglutinin (WGA)-Sepharose and were precipitated by concanavalin A, Wistaria floribunda hemagglutinin and WGA. Asialoglycoconjugate bound to concanavalin A-Sepharose and precipitated with concanavalin-A and W. floribunda hemagglutinin but not with WGA. Cells grown in the presence of fetal calf serum were agglutinated by WGA but not by peanut agglutinin. The reverse was true for cells grown without fetal calf serum. Neuraminidase-treated cells incorporated sialic acid or its 7-carbon analog, 5-acetamido-3,5-dideoxy-L-arabino-2-heptulosonic acid (AcNeu7) from sialylated compounds such as fetuin or sialyl-lactose but did not incorporate free sialic acid. Restoration of the WGA sialylreceptors in neuraminidase-treated cells, as determined by cell agglutination with WGA, was also obtained by incubation with fetuin or sialyl-lactose but not with free sialic acid. Moreover, restoration of agglutinability by WGA in neuraminidase-treated cells or cells grown in medium without fetal calf serum occurred equally well in energy-rich or energy-depleted cells. A transglycosilase reaction for sialic acid incorporation in T. cruzi epimastigotes is suggested.

Suppressive effect of ethanol on the expression of hepatic asialoglycoprotein receptors augmented by interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha.

Blood levels of inflammatory-related cytokines, including interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha, are elevated in patients with alcoholic liver diseases. We investigated the effects of these cytokines and ethanol on the expression of hepatic asialoglycoprotein receptors (AGPRs) in a human hepatoblastoma cell line, HepG2. An [125I]-asialo-orosomucoid binding assay showed significant increases in surface AGPR numbers in HepG2 cells by treatment with IL-1beta, IL-6, and TNF-alpha, to levels which were approximately 130% of the values in untreated control cells. However, the enhanced AGPR numbers induced by treatment with these cytokines were markedly suppressed, to 70%-80% of the number in the untreated cells, by treatment with ethanol. Immunological detection of AGPR with a specific antibody demonstrated that the modulation of surface AGPR numbers was correlated with the cellular expression levels of AGPR. These results suggest that, although IL-1beta, IL-6, and TNF-alpha stimulate the synthesis of hepatic AGPR, ethanol suppresses the expression of AGPR augmented by these cytokines. This leads to an increase in serum asialo-orosomucoid levels caused by the disordered catabolism mediated by AGPR in patients with alcoholic liver disease.

The nonerythropoietic asialoerythropoietin protects against neonatal hypoxia-ischemia as potently as erythropoietin.

Recently, erythropoietin (EPO) and the nonerythropoietic derivative asialoEPO have been linked to tissue protection in the nervous system. In this study, we tested their effects in a model of neonatal hypoxia-ischemia (HI) in 7-day-old rats (unilateral carotid ligation and exposure to 7.7% O(2) for 50 min). EPO (10 U/g body weight = 80 ng/g; n = 24), asialoEPO (80 ng/g; n = 23) or vehicle (phosphate-buffered saline with 0.1% human serum albumin; n = 24) was injected intraperitoneally 4 h before HI. Both drugs were protective, as judged by measuring the infarct volumes, neuropathological score and gross morphological score. The infarct volumes were significantly reduced by both EPO (52%) and asialoEPO (55%) treatment, even though the plasma levels of asialoEPO had dropped below the detection limit (1 pm) at the onset of HI, while those of EPO were in the nanomolar range. Thus, a brief trigger by asialoEPO before the insult appears to be sufficient for protection. Proteomics analysis after asialoEPO treatment alone (no HI) revealed at least one differentially up-regulated protein, synaptosome-associated protein of 25 kDa (SNAP-25). Activation (phosphorylation) of ERK was significantly reduced in asialoEPO-treated animals after HI. EPO and the nonerythropoietic asialoEPO both provided significant and equal neuroprotection when administered 4 h prior to HI in 7-day-old rats. The protection might be related to reduced ERK activation and up-regulation of SNAP-25.