Acidic Mammalian Chitinase Research Articles

Evaluation of AMCase and CHIT-1 expression in monocyte macrophages lineage

Acidic mammalian chitinase (AMCase) and chitotriosidase (CHIT-1) are two active chitinases expressed in humans. The chitinase activity of AMCase was found to be causative in allergic inflammation and its expression was found to be induced by interleukin-13. CHIT1-1 is expressed by phagocytic cells and extremely high levels are seen in lysosomal storage diseases. Despite that AMCase expression in the inflammation is under investigation, little is known regarding its regulation during macrophages' full maturation and polarization. In this study, we compared AMCase and CHIT-1 modulation during monocyte to macrophage transition and polarization. Gene expression analysis was investigated by real-time PCR from mRNA of human monocytes obtained from buffy coat of healthy volunteers, from mRNA of polarized to classically activated macrophages (or M1), obtained by interferon (IFN)-γ and lipopolysaccharide (LPS) treatment, and from mRNA of alternatively activated macrophages (or M2) obtained by interleukin (IL)-4 exposure. Our results showed that the expression of AMCase and CHIT-1 were differently modulated in HMMs at different stage of maturation. The behavior of these two active chitinase suggests that in the immune response their role is complementary.

Study on the Discovery of Novel Chitinase Inhibitors Based on Natural Products

Chitin, the second most abundant polysaccharide in nature, is a constituent of fungal cell walls, the exoskeletons of crustaceans and insects and the microfilarial sheaths of parasitic nematodes. Chitin has, so far, not been found in mammals. Accumulation of chitin by organisms is modulated by chitin synthase-mediated biosynthesis and by chitinase-mediated hydrolytic degradation. Thus, chitinases are expected to be specific targets for antifungal, insecticidal and antiparasitic agents. Paradoxically, while chitin does not exist in mammals, human chitinase family members, such as acidic mammalian chitinase, have recently been described, and offer significant potential for the treatment of asthma and other related diseases in humans. This review covers the development of two chitinase inhibitors of natural origin, Argifin and Argadin, isolated from the cultured broth of microorganisms in our laboratory. In particular, the practical total synthesis of these natural products and discovery methods that generate only highly-active compounds using a kinetic target (chitinase)-guided synthesis approach (termed in situ click chemistry) are described.

Effects of dexamethasone or methotrexate on chitinolytic activity in nasal polyps

Chitin is a recognition element for tissue infiltration by innate cells implicated in allergy and immunity. This process can be negatively regulated by vertebrate chitinases. Chitinolytic activity is significantly increased in nasal polyps (NPs) compared with that in normal turbinate mucosa. Dexamethasone (DEX) or methotrexate (MTX) is an effective treatment for chronic inflammatory diseases. The aim of this study was to investigate the effects of DEX or MTX on chitinolytic activity in organ-cultured NPs. NP tissues were cultured using an air-liquid interface culture model. Cultures were maintained for 24 hours in the absence or presence of DEX (10 or 100 micromolar) or MTX (10 or 100 micromolar). Acidic mammalian chitinase (AMCase) and chitotriosidase (ChT) activities in tissue samples were measured at a range of pH values by using the fluorogenic substrate 4-methylumbelliferyl-beta-(D)-N,N',N″-triacetyl-chitotriose. AMCase and ChT chitinolytic activities were significantly lower in NPs treated with 100 micromolar DEX or 100 micromolar MTX than that in fresh or untreated NPs. AMCase chitinolytic activity tended to be lower in DEX-treated polyps than in MTX-treated polyps, although the difference was not statistically significant. DEX or MTX reduced chitinolytic activity in NPs. We suggest that DEX or MTX may inhibit the growth of NPs via local regulation of NP chitinolytic activity.

Effects of an anthraquinone derivative from Rheum officinale Baill, emodin, on airway responses in a murine model of asthma

Emodin is a component from traditional Chinese herbal medicines. We focused on investigating whether emodin possesses distinct anti-inflammatory activity on a non-infectious mouse model of asthma, and we aimed to elucidate its involvement with the NF-κB pathway. BALB/c mice that were sensitized and challenged to ovalbumin were treated with emodin (40mg/kg) 1h before they were challenged with OVA. Our study demonstrated that emodin inhibited OVA-induced increases in eosinophil count; interleukin (IL)-4, IL-5, and IL-13 levels were recovered in bronchoalveolar lavage fluid and reduced serum levels of OVA-specific IgE, IgG, and IgG1. Histological studies demonstrated that emodin substantially inhibited OVA-induced eosinophilia in lung tissue and mucus hyper-secretion by goblet cells in the airway. Furthermore, pretreatment with emodin resulted in a significant reduction in mRNA expression of acidic mammalian chitinase (AMCase), chitinase 3-like protein 4 (Ym2) and Muc5ac in lung tissues and airway hyperresponsiveness to methacholine. These findings suggest that emodin may effectively delay the progression of airway inflammation and could be used as a therapy for patients with allergic airway inflammation.

Chitinase-like Proteins are Candidate Biomarkers for Sepsis-induced Acute Kidney Injury

Sepsis-induced acute kidney injury (AKI) is a frequent complication of critically ill patients and leads to high mortality rates. The specificity of currently available urinary biomarkers for AKI in the context of sepsis is questioned. This study aimed to discover urinary biomarkers for septic AKI by contemporary shotgun proteomics in a mouse model for sepsis and to validate these in individual urine samples of mice and human septic patients with and without AKI. At 48 h after uterine ligation and inoculation of Escherichia coli, aged mice (48 weeks) became septic. A subgroup developed AKI, defined by serum creatinine, blood urea nitrogen, and renal histology. Separate pools of urine from septic mice with and without AKI mice were collected during 12 h before and between 36-48 h after infection, and their proteome compositions were quantitatively compared. Candidate biomarkers were validated by Western blot analysis of urine, plasma, and renal tissue homogenates from individual mice, and a limited number of urine samples from human septic patients with and without AKI. Urinary neutrophil gelatinase-associated lipocalin, thioredoxin, gelsolin, chitinase 3-like protein 1 and -3 (CHI3L3) and acidic mammalian chitinase were the most distinctive candidate biomarkers selected for septic AKI. Both neutrophil gelatinase-associated lipocalin and thioredoxin were detected in urine of septic mice and increased with severity of AKI. Acidic mammalian chitinase was only present in urine of septic mice with AKI. Both urinary chitinase 3-like protein 1 and -3 were only detected in septic mice with severe AKI. The human homologue chitinase 3-like protein 1 was found to be more excreted in urine from septic patients with AKI than without. In summary, urinary chitinase 3-like protein 1 and -3 and acidic mammalian chitinase discriminated sepsis from sepsis-induced AKI in mice. Further studies of human chitinase proteins are likely to lead to additional insights in septic AKI.

Role of Chitotriosidase (Chitinase 1) Under Normal and Disease Conditions

Mammalian chitinases belong to the glycosyl hydrolase 18 family based on structural homology and the family includes a large number of bacterial and eukaryotic chitinases. Among the mammalian chitinases, chitotriosidase (CHIT1) and acidic mammalian chitinase (AMCase) are capable of hydrolyzing the β-(1, 4)-linkage between the adjacent N-acetyl glucosamine residues of chitin. CHIT1 is one of the most abundantly secreted proteins, being mainly produced by activated macrophages and epithelial cells. CHIT1 plays a pivotal role in the context of infectious disease including malaria and fungi infections as a host defense towards chitin in pathogen's cell structure and as a diagnostic marker of disease. In contrast, CHI1 released by activated Kupffer cells in liver could induce hepatic fibrosis and cirrhosis. Increased serum levels of CHIT1 were observed in patients with many disorders, including Gaucher's disease, bronchial asthma, and atherosclerosis. Therefore, CHIT1 seems to have dual (regulatory and pathogenic) roles depending on the disease and producing cell types during the inflammatory conditions.

Acidic Mammalian Chitinase Is Not a Critical Target for Allergic Airway Disease

The expression of acidic mammalian chitinase (AMCase) is associated with Th2-driven respiratory disorders. To investigate the potentially pathological role of AMCase in allergic airway disease (AAD), we sensitized and challenged mice with ovalbumin or a combination of house dust mite (HDM) plus cockroach allergen. These mice were treated or not treated with small molecule inhibitors of AMCase, which significantly reduced allergen-induced chitinolytic activity in the airways, but exerted no apparent effect on pulmonary inflammation per se. Transgenic and AMCase-deficient mice were also submitted to protocols of allergen sensitization and challenge, yet we found little or no difference in the pattern of AAD between mutant mice and wild-type (WT) control mice. In a separate model, where mice were challenged only with intratracheal instillations of HDM without adjuvant, total bronchoalveolar lavage (BAL) cellularity, inflammatory infiltrates in lung tissues, and lung mechanics remained comparable between AMCase-deficient mice and WT control mice. However BAL neutrophil and lymphocyte counts were significantly increased in AMCase-deficient mice, whereas concentrations in BAL of IL-13 were significantly decreased compared with WT control mice. These results indicate that, although exposure to allergen stimulates the expression of AMCase and increased chitinolytic activity in murine airways, the overexpression or inhibition of AMCase exerts only a subtle impact on AAD. Conversely, the increased numbers of neutrophils and lymphocytes in BAL and the decreased concentrations of IL-13 in AMCase-deficient mice challenged intratracheally with HDM indicate that AMCase contributes to the Th1/Th2 balance in the lungs. This finding may be of particular relevance to patients with asthma and increased airway neutrophilia.

Genetic association between human chitinases and lung function in COPD.

Two primary chitinases have been identified in humans--acid mammalian chitinase (AMCase) and chitotriosidase (CHIT1). Mammalian chitinases have been observed to affect the host's immune response. The aim of this study was to test for association between genetic variation in the chitinases and phenotypes related to chronic obstructive pulmonary disease (COPD). Polymorphisms in the chitinase genes were selected based on previous associations with respiratory diseases. Polymorphisms that were associated with lung function level or rate of decline in the Lung Health Study (LHS) cohort were analyzed for association with COPD affection status in four other COPD case-control populations. Chitinase activity and protein levels were also related to genotypes. In the caucasian LHS population, the baseline forced expiratory volume in one second (FEV(1)) was significantly different between the AA and GG genotypic groups of the AMCase rs3818822 polymorphism. Subjects with the GG genotype had higher AMCase protein and chitinase activity compared with AA homozygotes. For CHIT1 rs2494303, a significant association was observed between rate of decline in FEV(1) and the different genotypes. In the African American LHS population, CHIT1 rs2494303 and AMCase G339T genotypes were associated with rate of decline in FEV(1). Although a significant effect of chitinase gene alleles was found on lung function level and decline in the LHS, we were unable to replicate the associations with COPD affection status in the other COPD study groups.

Human Chitotriosidase-Catalyzed Hydrolysis of Chitosan

Chitotriosidase (HCHT) is one of two family 18 chitinases produced by humans, the other being acidic mammalian chitinase (AMCase). The enzyme is thought to be part of the human defense mechanism against fungal parasites, but its precise role and the details of its enzymatic properties have not yet been fully unraveled. We have studied the properties of HCHT by analyzing how the enzyme acts on high-molecular weight chitosans, soluble copolymers of β-1,4-linked N-acetylglucosamine (GlcNAc, A), and glucosamine (GlcN, D). Using methods for in-depth studies of the chitinolytic machinery of bacterial family 18 enzymes, we show that HCHT degrades chitosan primarily via an endoprocessive mechanism, as would be expected on the basis of the structural features of its substrate-binding cleft. The preferences of HCHT subsites for acetylated versus nonacetylated sugars were assessed by sequence analysis of obtained oligomeric products showing a very strong, absolute, and a relative weak preference for an acetylated unit in the -2, -1, and +1 subsites, respectively. The latter information is important for the design of inhibitors that are specific for the human chitinases and also provides insight into what kind of products may be formed in vivo upon administration of chitosan-containing medicines or food products.

Overexpression of chitinase 3-like 1/YKL-40 in lung-specific IL-18-transgenic mice, smokers and COPD.

We analyzed the lung mRNA expression profiles of a murine model of COPD developed using a lung-specific IL-18-transgenic mouse. In this transgenic mouse, the expression of 608 genes was found to vary more than 2-fold in comparison with control WT mice, and was clustered into 4 groups. The expression of 140 genes was constitutively increased at all ages, 215 genes increased gradually with aging, 171 genes decreased gradually with aging, and 82 genes decreased temporarily at 9 weeks of age. Interestingly, the levels of mRNA for the chitinase-related genes chitinase 3-like 1 (Chi3l1), Chi3l3, and acidic mammalian chitinase (AMCase) were significantly higher in the lungs of transgenic mice than in control mice. The level of Chi3l1 protein increased significantly with aging in the lungs and sera of IL-18 transgenic, but not WT mice. Previous studies have suggested Chi3l3 and AMCase are IL-13-driven chitinase-like proteins. However, IL-13 gene deletion did not reduce the level of Chi3l1 protein in the lungs of IL-18 transgenic mice. Based on our murine model gene expression data, we analyzed the protein level of YKL-40, the human homolog of Chi3l1, in sera of smokers and COPD patients. Sixteen COPD patients had undergone high resolution computed tomography (HRCT) examination. Emphysema was assessed by using a density mask with a cutoff of −950 Hounsfield units to calculate the low-attenuation area percentage (LAA%). We observed significantly higher serum levels in samples from 28 smokers and 45 COPD patients compared to 30 non-smokers. In COPD patients, there was a significant negative correlation between serum level of YKL-40 and %FEV1. Moreover, there was a significant positive correlation between the serum levels of YKL-40 and LAA% in COPD patients. Thus our results suggest that chitinase-related genes may play an important role in establishing pulmonary inflammation and emphysematous changes in smokers and COPD patients.

Fungal Chitin from Asthma-Associated Home Environments Induces Eosinophilic Lung Infiltration

Development of asthma and allergic inflammation involves innate immunity, but the environmental contributions remain incompletely defined. Analysis of dust collected from the homes of asthmatic individuals revealed that the polysaccharide chitin is environmentally widespread and associated with β-glucans, possibly from ubiquitous fungi. Cell wall preparations of Aspergillus isolated from house dust induced robust recruitment of eosinophils into mouse lung, an effect that was attenuated by enzymatic degradation of cell wall chitin and β-glucans. Mice expressing constitutively active acidic mammalian chitinase in the lungs demonstrated a significant reduction in eosinophil infiltration after fungal challenge. Conversely, chitinase inhibition prolonged the duration of tissue eosinophilia. Thus, fungal chitin derived from home environments associated with asthma induces eosinophilic allergic inflammation in the lung, and mammalian chitinases, including acidic mammalian chitinase, limit this process.

Expression of Chitinases in Hypertrophied Adenoids of Children

Chronic rhinosinusitis (CRS), otitis media with effusion (OME), and allergic rhinitis (AR) are common conditions that have been associated with hypertrophied adenoids in children, and adenoidectomy is clinically recommended. The investigators assayed the expression level and site of acidic mammalian chitinase (AMCase) and chitotriosidase (ChT) in hypertrophied adenoids of children to determine the expression levels of 2 chitinases in relation to CRS, OME, and AR. A prospective cohort study. A tertiary care facility. Hypertrophied adenoids from 41 children were harvested during adenoidectomy. Medical records were reviewed and the subjects were grouped according to the presence of CRS, OME, and AR. Messenger RNA (mRNA) and protein expression of AMCase and ChT in adenoid tissues was assessed by reverse transcription polymerase chain reaction and Western blotting. Immunohistochemical staining revealed the sites of AMCase and ChT expression. mRNA and protein of AMCase and ChT were present in adenoids of all subjects. CRS was a significant variable in AMCase mRNA and protein expression. CRS, OME, and AR were significant variables in ChT mRNA and protein expression. Both AMCase and ChT were expressed in histiocytes and vascular endothelial cells of adenoid tissues. The findings suggest that chitin-containing pathogens or dysregulated immune responses to them in the hypertrophied adenoids of children could be factors contributing to CRS, OME, and AR via AMCase or ChT overexpression.

Analyzing Airway Inflammation with Chemical Biology: Dissection of Acidic Mammalian Chitinase Function with a Selective Drug-like Inhibitor

SummaryAcidic mammalian chitinase (AMCase) is produced in the lung during allergic inflammation and asthma, and inhibition of enzymatic activity has been considered as a therapeutic strategy. However, most chitinase inhibitors are nonselective, additionally inhibiting chitotriosidase activity. Here, we describe bisdionin F, a competitive AMCase inhibitor with 20-fold selectivity for AMCase over chitotriosidase, designed by utilizing the AMCase crystal structure and dicaffeine scaffold. In a murine model of allergic inflammation, bisdionin F-treatment attenuated chitinase activity and alleviated the primary features of allergic inflammation including eosinophilia. However, selective AMCase inhibition by bisdionin F also caused dramatic and unexpected neutrophilia in the lungs. This class of inhibitor will be a powerful tool to dissect the functions of mammalian chitinases in disease and represents a synthetically accessible scaffold to optimize inhibitory properties in terms of airway inflammation.

The effects of an insertion in the 5′UTR of the AMCase on gene expression and pulmonary functions

Studies regarding the physiological role of acidic mammalian chitinase (AMCase) and the effects of its genetic variants on asthma have produced conflicting results. We aimed to determine the genetic variants in the AMCase gene that could regulate the gene expression and thus influence disease severity. Genetic variants of the AMCase gene were determined by sequencing of asthmatics and healthy controls in up to -1kb in the promoter region and exon 1 and 2. In an association study, a population of asthmatic (n=504) and healthy Turkish children (n=188) were genotyped for the observed SNPs. A replication study was performed in a North American adult population of patients with mild (n=317) and severe (n=145) asthma. The functional properties of the insertion were determined by promoter reporter assay, electromobility shift assay and transcription factor ELISA experiments. Of the identified SNPs, only a ten base pair insertion (CAATCTAGGC) in the 5'UTR region of exon 2 was significantly associated with lower FEV(1) (β=-14.63 SE=6.241, P=0.019) in Turkish children with asthma. However, in the adult population, the same insertion showed a trend toward higher FEV(1). The insertion was shown to have enhancer activity and the mutant probe possessing the insertion had higher binding affinity for the nuclear extracts. Our study shows that a ten base pair insertion in the 5'UTR region of AMCase gene may modify gene expression and thus may affect the severity of asthma. However, its effects appear to be different in different populations.

A positive feedback mechanism in the regulation of mammalian chitinase responses (56.29)

Abstract Immunosuppressed patients are highly susceptible to invasive fungal infections (IFI) such as invasive pulmonary aspergillosis, which is predominantly caused by the fungus Aspergillus fumigatus. An important component of the fungal cell wall is chitin, a polymer of N-acetyl-D-glucosamine (GlcNAc). Chitin is not produced by humans, however, the chitin degrading enzymes (chitinases) chitotriosidase (Chit-1) and acidic mammalian chitinase (AMCase) are. Chitinase is predominantly produced by activated macrophages, and may possibly aid in the defense against chitin-containing pathogens. We show that serum and bronchoalveolar lavage (BAL) chitinase levels are increased in patients with IFI, and in mice after pulmonary exposure to A. fumigatus conidia. Several different stimuli, including stimulation with chitin can lead to chitinase responses. In vitro stimulation of U937 human monocytes and RAW mouse macrophages with either chitin-particles (7-12 µm) or GlcNAc increased secreted and intracellular chitinase activity, the accumulation of intracellular Chit-1, and significantly increased the mRNA levels of Chit-1 and AMCase. Accordingly, we hypothesize that Chit-1 expression is regulated through a positive feedback mechanism involving the degradation of chitin to GlcNAc by host chitinases and GlcNAc recognition that in turn upregulates chitinase expression. This potential feedback mechanism of chitinase regulation may have utility in the diagnosis of IFIs.

Role of Eotaxin‐3 in Chronic Rhinosinusitis with Nasal Polyps

Chronic rhinosinusitis with nasal polyps (CRSwNP) is connected to a chronic inflammatory process with the activation of different immune components. Eotaxin-3, acidic mammalian chitinase (AMCase), and interleukin (IL)–13 as focal immune factors have been detected in CRSwNP, but their importance and relationship are still being investigated. This study aims to detect the expression of AMCase, IL-13, and eotaxin-3 in the mRNA level and investigate their roles and relationships in CRSwNP. Twenty-four subjects with eosinophilic CRSwNP and 11 controls were included in the study. Tissues were obtained by endoscopic sinus surgery. Target genes were detected by real-time reverse transcription–polymerase chain reaction. AMCase, eotaxin-3, and IL-13 were detected in all CRSwNP and controls. The expression of AMCase, eotaxin-3, IL-13, and mRNA was significantly higher in patients with CRSwNP than in the control group. There was a significantly positive correlation not only between AMCase and eotaxin-3 but also between IL-13 and eotaxin-3 in CRSwNP. Increased AMCase, IL-13, and eotaxin-3 might lead to the Th2 inflammatory cascade reaction.

Role of Chitin and Chitinase/Chitinase-Like Proteins in Inflammation, Tissue Remodeling, and Injury

The 18 glycosyl hydrolase family of chitinases is an ancient gene family that is widely expressed from prokaryotes to eukaryotes. In mammals, despite the absence of endogenous chitin, a number of chitinases and chitinase-like proteins (C/CLPs) have been identified. However, their roles have only recently begun to be elucidated. Acidic mammalian chitinase (AMCase) inhibits chitin-induced innate inflammation; augments chitin-free, allergen-induced Th2 inflammation; and mediates effector functions of IL-13. The CLPs BRP-39/YKL-40 (also termed chitinase 3-like 1) inhibit oxidant-induced lung injury, augments adaptive Th2 immunity, regulates apoptosis, stimulates alternative macrophage activation, and contributes to fibrosis and wound healing. In accord with these findings, levels of YKL-40 in the lung and serum are increased in asthma and other inflammatory and remodeling disorders and often correlate with disease severity. Our understanding of the roles of C/CLPs in inflammation, tissue remodeling, and tissue injury in health and disease is reviewed below.

Acidic Mammalian Chitinase and the Eye: Implications for Ocular Inflammatory Diseases

Chitinases have an important role in the defense of organisms against chitin-containing parasites. An acidic mammalian chitinase (AMCase) has been detected in epithelial cells in lung tissue samples taken from patients with asthma as well as in conjunctival epithelium of patients with inflammatory ocular diseases. Particularly, elevated AMCase activity has been observed in ocular tissues of patients with vernal keratoconjunctivitis, seasonal allergic conjunctivitis, and in patients affected by dry eye syndrome. This enzyme is induced via a TH2-specific, IL-13-dependent pathway. AMCase may thus be a key mediator of IL-13-induced responses in TH2-driven inflammatory ocular diseases.

Acidic mammalian chitinase (AMCase): A new target for ocular diseases

Acidic mammalian chitinase (AMCase): A new target for ocular diseases

Acidic mammalian chitinase (AMCase): A new target for ocular diseases

Acidic mammalian chitinase (AMCase): A new target for ocular diseases