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ABSTRACT Murine macrophages are activated by interferon-γ (IFN-γ) and/or Toll-like receptor (TLR) agonists such as bacterial endotoxin (lipopolysaccharide [LPS]) to express an inflammatory (M1) phenotype characterized by the expression of nitric oxide synthase-2 (iNOS) and inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-12. In contrast, Th2 cytokines IL-4 and IL-13 activate macrophages by inducing the expression of arginase-1 and the anti-inflammatory cytokine IL-10 in an IL-4 receptor-α (IL-4Rα)-dependent manner. Macrophages activated in this way are designated as “alternatively activated” (M2a) macrophages. We have shown previously that adenosine A 2A receptor (A 2A R) agonists act synergistically with TLR2, TLR4, TLR7, and TLR9 agonists to switch macrophages into an “M2-like” phenotype that we have termed “M2d.” Adenosine signaling suppresses the TLR-dependent expression of TNF-α, IL-12, IFN-γ, and several other inflammatory cytokines by macrophages and induces the expression of vascular endothelial growth factor (VEGF) and IL-10. We show here using mice lacking a functional IL-4Rα gene (IL-4Rα −/− mice) that this adenosine-mediated switch does not require IL-4Rα-dependent signaling. M2d macrophages express high levels of VEGF, IL-10, and iNOS, low levels of TNF-α and IL-12, and mildly elevated levels of arginase-1. In contrast, M2d macrophages do not express Ym1, Fizz1 (RELM-α), or CD206 at levels greater than those induced by LPS, and dectin-1 expression is suppressed. The use of these markers in vivo to identify “M2” macrophages thus provides an incomplete picture of macrophage functional status and should be viewed with caution.

Sandhoff disease (SD) is caused by deficiency of N-acetyl-β-hexosaminidase (Hex) resulting in pathological accumulation of GM2 ganglioside in lysosomes of the central nervous system (CNS) and progressive neurodegeneration. Currently, there is no treatment for SD, which often results in death by the age of five years. Adeno-associated virus (AAV) gene therapy achieved global CNS Hex restoration and widespread normalization of storage in the SD mouse model. Using a similar treatment approach, we sought to translate the outcome in mice to the feline SD model as an important step toward human clinical trials. Sixteen weeks after four intracranial injections of AAVrh8 vectors, Hex activity was restored to above normal levels throughout the entire CNS and in cerebrospinal fluid, despite a humoral immune response to the vector. In accordance with significant normalization of a secondary lysosomal biomarker, ganglioside storage was substantially improved, but not completely cleared. At the study endpoint, 5-month-old AAV-treated SD cats had preserved neurological function and gait compared with untreated animals (humane endpoint, 4.4±0.6 months) demonstrating clinical benefit from AAV treatment. Translation of widespread biochemical disease correction from the mouse to the feline SD model provides optimism for treatment of the larger human CNS with minimal modification of approach.

Rab44 is a large Rab GTPase containing a Rab GTPase domain and some additional N‐terminal domains. We recently used Rab44‐deficient mice to demonstrate that Rab44 regulates granule exocytosis in mast cells and IgE‐mediated anaphylaxis. In mouse mast cells, Rab44 is expressed as two isoforms, namely, the long and short forms; however, the characteristics of these two isoforms remain unknown. Here, we investigated secretion and localization of the human long Rab44 isoform and the two mouse isoforms and their mutants expressed in rat basophilic leukemia (RBL)‐2H3 cells. Expression of the human long isoform and both mouse isoforms caused an increase in β‐hexosaminidase secretion. Confocal and quantitative analyses showed that both human and mouse long isoforms localized mainly to lysosomes while the mouse short isoform localized mainly to the ER. Live imaging with LysoTracker indicated that the size and number of LysoTracker‐positive vesicles were altered by the various mutants. Ionomycin treatment partially altered localization of both long isoforms to the plasma membrane and cytosol, whereas it had little effect on colocalization of the short isoform with lysosomes. Mechanistically, both human and mouse Rab44 proteins interacted with vesicle‐associated membrane protein 8 (VAMP8), a v‐SNARE protein. Therefore, Rab44 isoforms similarly promote lysosomal exocytosis, but exhibit differential localization in mast cells. We investigated the difference in function and localization of the human and mouse Rab44 isoforms and their mutants. Expression of the human and mouse long and short isoforms increased β‐hexosaminidase secretion in rat basophilic leukemia‐2H3 cells. However, both human and mouse long isoforms localized mainly to lysosomes while the mouse short isoform localized mainly to the ER.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic lung disorder of unknown cause. Several studies suggest an association between Epstein-Barr virus pulmonary infection and the development of IPF. To determine whether reduction of gamma-herpesvirus reactivation from latency would alter progressive lung fibrogenesis in an animal model of virus-induced pulmonary fibrosis. IFN-gamma receptor-deficient (IFN-gammaR(-/-)) mice infected intranasally with murine gamma-herpesvirus 68 (MHV68) develop lung fibrosis that progresses for up to at least 180 days after initial infection. Viral replication during the chronic phase of infection was controlled by two methods: the administration of cidofovir, an antiviral drug effective at clearing lytic but not latent virus, and by using a mutant gamma-herpesvirus defective in virus reactivation from latency. Ten percent of the asymptomatic MHV68-infected animals that received antiviral treatment beginning on Day 45 postinfection had severe pulmonary fibrosis compared with 40% of the control saline-treated animals. Absence of severe fibrosis was also observed in IFN-gammaR(-/-) mice infected with the defective reactivation mutant MHV68 v-cyclin stop. Decreased fibrosis was associated with lower levels of transforming growth factor-beta, vascular endothelial growth factor, and markers of macrophage alternative activation. When antiviral treatment was administered on Day 60 in symptomatic animals, survival improved from 20 to 80% compared with untreated symptomatic animals, but lung fibrosis persisted in 60% of the mice. MHV68-induced fibrosis is a result of viral lytic replication during chronic lung herpesvirus infection in mice. We speculate that antiviral therapy might help to control lung fibrosis in humans with IPF and associated herpesvirus infection.

CD1 proteins present various glycolipid antigens to T cells, but the cellular mechanisms that control which particular glycolipids generate T cell responses are not understood. We show here that T cell recognition of glucose monomycolate antigens with long (C 80 ) alkyl chains involves the delivery of CD1b proteins and antigens to late endosomes in a process that takes several hours. In contrast, analogs of the same antigen with shorter (C 32 ) alkyl chains are rapidly, but inefficiently, presented by cell surface CD1b proteins. Dendritic cells (DCs) preferentially present long-chain glycolipids, which results, in part, from their rapid internalization and selective delivery of antigens to endosomal compartments. Nonprofessional antigen-presenting cells, however, preferentially present short-chain glycolipids because of their lack of prominent endosomal presentation pathways. Because long alkyl chain length distinguishes certain microbial glycolipids from common mammalian glycolipids, these findings suggest that DCs use a specialized endosomal-loading pathway to promote preferential recognition of glycolipids with a more intrinsically foreign structure.

This study evaluated whether GM 2 ganglioside storage is necessary for neurodegeneration and neuroinflammation by performing β-hexosaminidase rescue experiments in neurons of HexB −/− mice. We developed a novel mouse model, whereby the expression of the human HEXB gene was targeted to neurons of HexB −/− mice by the Thy1 promoter. Despite β-hexosaminidase restoration in neurons was sufficient in rescuing HexB −/− mice from GM 2 neuronal storage and neurodegeneration, brain inflammation persisted, including the presence of large numbers of reactive microglia/macrophages due to persisting GM 2 presence in this cell type. In conclusion, our results suggest that neuroinflammation is not sufficient to elicit neurodegeneration as long as neuronal function is restored.

Ym is one of the chitinase family proteins, which are widely distributed in mammalian bodies and can bind glycosaminoglycans such as heparin/heparan sulfate. Ym1 is a macrophage protein produced in parasitic infections, while its isoform, Ym2, is upregulated in lung under allergic conditions. In the present study, we revealed the distinct cellular expression of Ym1 and Ym2 in normal mice by in situ hybridization and immunohistochemistry. Ym1 was principally expressed in the lung, spleen, and bone marrow, while Ym2 was found in the stomach. Ym1-expressing cells in the lung were alveolar macrophages, and the immunoreactivity for Ym1 was localized in rough endoplasmic reticulum. In the spleen, Ym1-expressing cells gathered in the red pulp and were electron microscopically identified as immature neutrophils. In the bone marrow, immature neutrophils were intensely immunoreactive, but lost this immunoreactivity with maturation. Moreover, needle-shaped crystals in the cytoplasm of macrophages, which formed erythroblastic islands, also showed intense Ym1 immunoreactivity. Ym2 expression was restricted to the stratified squamous epithelium in the junctional region between forestomach and glandular stomach. The function of Ym1 and Ym2 is still unclear; however, the distinct cellular localization under normal conditions suggests their important roles in hematopoiesis, tissue remodeling, or immune responses as an endogenous lectin.

Glucosamine (GlcN)-induced insulin resistance is associated with an increase in O-linked-N-acetylglucosaminylated modified proteins (O-GlcNAcylated proteins). The role played by O-GlcNAc-selective-N-acetyl-β-D-glucosaminidase (O-GlcNAcase), which removes O-N-acetyl-glucosamine residues from O-GlcNAcylated proteins, has not yet been demonstrated. We investigated whether GlcN-induced whole-body insulin resistance is related to tissue O-GlcNAcase activity and mRNA expression. GlcN (30 µmol/kg/min) or physiological saline (control) was intravenously infused into Sprague-Dawley rats for 2 h. After GlcN treatment, rats were subjected to the following: intravenous glucose tolerance test, insulin tolerance test or removal of the liver, muscle and pancreas. GlcN was found to provoke hyperglycemia compared to control (8.6 ± 0.41 vs. 4.82 ± 0.17 mM, p < 0.001). The insulin resistance index (HOMA-IR) increased (15.76 ± 1.47 vs. 10.14 ± 1.41, p < 0.001) and the β-cell function index (HOMA-β) diminished (182.69 ± 22.37 vs. 592.01 ± 103, p < 0.001). Liver glucose concentration was higher in the GlcN group than in the control group (0.37 ± 0.04 vs. 0.24 ± 0.038 mmol/g dry weight, p < 0.001). Insulin release index (insulin/glucose) was less in the GlcN group than in the control (2.2 ± 0.1 vs. 8 ± 0.8 at 120 min, p < 0.001). In the GlcN group, muscle O-GlcNAcase activity diminished (0.28 ± 0.019 vs. 0.36 ± 0.018 nmol of p-nitrophenyl/mg protein/min, p < 0.001), and K m increased (1.51 ± 0.11 vs. 1.12 ± 0.1 mM, p < 0.001) compared to the control. In the GlcN group, O-GlcNAcase activity/mRNA expression was altered (0.6 ± 0.07 vs. 1 ± 0.09 of control, p < 0.05). In conclusion, O-GlcNAcase activity is posttranslationally inhibited during GlcN-induced insulin resistance.

Background: N-acetyl-β-hexosaminidase (β-hex) is a lysosomal hydrolase, which is selectively secreted into the extracellular space by inflammatory cells. The aim of our study was to assess the activity of β-hex in the plasma of asthmatic patients, and to establish whether it correlates with asthma severity and airway inflammation. Methods: The study was conducted in a group of 46 asthmatic patients and 13 healthy volunteers. All study participants underwent analysis of exhaled nitric oxide and flow-volume spirometry. β-hex activity, peripheral blood eosinophils, total serum IgE and eosinophil cationic protein were analyzed in blood samples from all asthmatic patients and healthy volunteers. Results: β-hex activity was significantly higher in patients with severe or moderate asthma compared with healthy volunteers and was positively correlated with exhaled nitric oxide levels and serum eosinophil cationic protein in these groups of patients. There was no correlation between β-hex activity and forced expiratory volume in 1 s, blood eosinophil count or total serum IgE in these groups of asthmatics. Conclusions: Our results suggest that β-hex could take part in airway inflammation and remodeling in asthma. Our study is the first report in which the elevated activity of β-hex in subjects with asthma has been observed. However, more studies are needed to establish the precise role of this enzyme in asthma in humans.

Mast cells play an important role in allergic inflammation by releasing various bioactive mediators. The function of mast cells is enhanced by various stimuli, partly due to the induction of specific genes and their products. Although many inducible genes have been identified, a significant number of genes remain to be identified. Therefore, this study used PCR‐selected cDNA subtraction to establish the profile of induced genes in the connective tissue (CT) type‐like mast cells derived from bone marrow cells cultured in the presence of IL‐4 and stem cell factor. Two hundred and fifty cDNA clones were obtained from the CT type‐like mast cells by PCR‐selected cDNA subtraction. Among them, Ym1/2, a chitinase‐like protein, is one of the most abundantly induced genes. Ym1 is produced by activated macrophages in a parasitic infection, whereas its isotype, Ym2, is highly upregulated in allergic lung disease. In order to differentiate which isotype is expressed in bone marrow cells, specific primers for bone marrow‐derived mast cells (BMMC), and CT type‐like mast cells were used for RT‐PCR. The results showed that Ym1 was constitutively expressed in bone marrow cells and gradually decreased in the presence of IL‐3, whereas Ym2 was induced only in the presence of IL‐4. CT type‐like mast cells from bone marrow cells expressed Ym1 throughout the culture period and Ym2 was induced only by the addition of IL‐4 into BMMC, indicating that IL‐4 is essential for the expression of Ym1/2 genes.