Chitosan leads to downregulation of YKL-40 and inflammasome activation in human macrophages Steinunn Gudmundsdottir,1 Ramona Lieder,2,3 Olafur E. Sigurjonsson,2,3,4 Petur H. Petersen1,4 1
Faculty of Medicine, School of Health Sciences, Department of Anatomy, Biomedical Center, University of Iceland, Reykjavik 101, Iceland 2 The Blood Bank, Landspitali University Hospital, Snorrabraut 60, Reykjavik 105, Iceland 3 School of Science and Engineering, Reykjavik University, Menntavegur 1, Reykjavik 101, Iceland 4 Biomedical Center, University of Iceland, Reykjavik 101, Iceland Received 23 September 2014; revised 18 December 2014; accepted 5 February 2015 Published online 27 February 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35417 Abstract: Chitosan, the deacetylated derivative of chitin, is used as biomaterial in diverse settings. It is also found on pathogens and can be proinflammatory. Shorter derivatives of chitosan can be generated chemically or enzymatically, chitosan oligosaccharides (ChOS). There is variation in the chemical composition of ChOS, including size distribution, but in general, they have been described as inert or antiinflammatory. Active human chitinases can cleave chitin and chitosan, while inactive chitinases bind both but do not cleave. Both active and inactive chitinases have important roles in the immune response. The inactive chitinase YKL-40 is expressed highly during inflammation and has been proposed as a marker of poor prognosis. YKL-40 acts as a negative regulator of the inflammasome and as a positive regulator of angiogenesis. Levels of YKL-40 can therefore reg-
ulate levels of inflammation, the extent of angiogenesis, and the process of inflammation resolution. This study shows that chitosan leads to reduced secretion of YKL-40 by primary human macrophages and that this is concomitant with inflammasome activation. This was most pronounced with a highly deacetylated ChOS. No effect on the secretion of the active chitinase Chit-1 was detected. Smaller and more acetylated ChOS did not affect YKL-40 levels nor inflammasome activation. We conclude that this effect on the levels of YKL40 is a part of the proinflammatory mechanisms of chitosan C 2015 Wiley Periodicals, Inc. J Biomed Mater and its derivatives. V Res Part A: 103A: 2778–2785, 2015.
Key Words: chitin, chitosan, inflammation, chitinase, YKL-40
How to cite this article: Gudmundsdottir S, Lieder R, Sigurjonsson OE, Petersen PH. 2015. Chitosan leads to downregulation of YKL-40 and inflammasome activation in human macrophages. J Biomed Mater Res Part A 2015:103A:2778–2785.
INTRODUCTION
The polysaccharide chitin, the polymer of Nacetylglucosamine, is found widely in nature, including as a part of various human pathogens. Deacetylation of chitin produces chitosan, which has increased water-solubility under acidic conditions.1,2 Chemically modified chitosan in various formulations shows potential as biomaterial, drug carrier, wound dressing, and transfection agent.3,4 Chitosan can be further cleaved enzymatically or chemically to generate smaller chitosan oligosaccharides (ChOS), which show increased solubility. ChOS, which are of variable length and different degrees of deacetylation, are more resistant to further degradation.5 ChOS are able to bind to chitin binding proteins, and can act as inhibitors of chitinases.6–8 While mammalian tissues do not contain chitin or chitosan, mammalian cells can express both active and inactive extracellular chitinases, the latter being able to bind, but not cleave chitin.9,10 Both take an active part in the immune
response,11,12 and likely play a special role in the response against pathogens which contain chitin or chitosan.13–15 Active chitinases, such as Chitotriosidase-1 (Chit-1) cleave chitin and chitin derivatives and can inhibit fungal growth.16 Inactive chitinases, of which YKL-40 (also known as CHI3L1 and BRP-39) is best characterized,17 also play a role in tissue repair, tissue remodeling11,18 and angiogenesis.19 Chit-1 and YKL-40, which are the focus of this study, are secreted by macrophages, and are highly upregulated in monocyte to macrophage differentiation.20,21 YKL-40 is upregulated in a large variety of pathological conditions and can be a marker of poor prognosis.22,23 Extracellular YKL-40 has been shown to play a major role in inhibiting the inflammasome, which leads to a reduction in the secretion of the proinflammatory cytokine IL-1b.24 YKL-40 could therefore play an important role in regulation and resolution of inflammation during infection, possibly in concert with changes in activation pattern of macrophages, that is, from classically activated
Correspondence to: P. H. Petersen; e-mail: [email protected] 1354-845-5346 Contract grant sponsor: Landspitali University Hospital Research Fund Contract grant sponsor: Icelandic Student Innovation Fund; contract grant number: 1323410091
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macrophages expressing YKL-40, to alternatively activated macrophages with reduced YKL-40 expression.25 However, little is known about the regulation of YKL-40. As chitosan is present on important human pathogens26 and its cleavage by Chit-1 leads to formation of ChOS, it is important to establish whether chitosan and ChOS have a direct effect on the expression of chitinases, both active and inactive. It has been suggested that ChOS, derived from degradation of chitosan in vivo, induces the expression of Chit-1 in THP-1 cells,27 which could be considered as positive feedback. Chitosan is used in diverse settings as biomaterial and has immunomodulating effects, that is, can act either antiinflammatory28,29 or proinflammatory,30 depending on the chemical composition of the chitosan and the experimental settings. This is further complicated by the in vivo degradation of chitosan into ChOS. ChOS also have diverse potential as biomaterials, such as scaffolds in tissue engineering, pharmaceuticals, vectors for gene delivery, in wound healing and as antifungal agents,3,5 and have been shown in general, to act as anti-inflammatory agents.31–33 The goal of this study was to test in primary human macrophages, one of the principal cell type of the immune response, whether chitosan and two different ChOS species had an effect on the secretion of Chit-1 and YKL-40 and whether they affected the activation of the inflammasome.
MATERIALS AND METHODS
Cell culture The THP-1 cell line34 was obtained from ATCC (Manassas, Virginia) and cultured in RPMI 1640 (Cat. no. 52400–025) supplemented with 10% FBS (Cat. no. 10270-106), 100 U/ mL Penicillin and 100 mg/mL Streptomycin (Cat. no. 15140122, Gibco, Carlsbad, CA). THP-1 cells (5 3 105 cells/mL) were differentiated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA, Cat. no. 524400, Calbiochem, San Diego, CA) in RPMI 1640 supplemented with 2% FBS for 2 days, followed by 2 days in PMA free media.35 Primary monocytes were isolated from buffy coat of healthy human volunteers with Ficoll-PaqueTM (Cat. no. 171440-02) density gradient centrifugation and MACS CD141 magnetic beads (Cat. no. 130-050-201, Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were seeded at 7.5 3 105 monocytes/mL in RPMI 1640 Glutamax (Cat. no. 61870, Gibco) medium containing 10% pooled normal human serum and 100 U/mL and 100 mg/mL Pen/Strep respectively (Cat. no. 10378016, Gibco). After 3 days, half of the medium was exchanged for fresh medium. On day 6, cells were washed with PBS (Cat. no. 10010-056, Gibco) and fresh medium added. Cells were stimulated with test substances in serum free medium on day 9; 24 h after stimulation the medium was collected for western blot and ELISA analysis and the cells were stained with crystal violet solution. Cells were fixed with 4% formaldehyde in PBS (Cat. no. 252549, Sigma-Aldrich), washed with PBS and stained with crystal violet solution (PBS w/10% v/v ethanol, 0.1% w/v crystal violet) for 20 min. Then, cells were washed three times with dH2O and relative cell numbers deter-
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mined by dissolving the dye with 30% acetic acid and measuring optical density at 570 nm. CHEMICALS
TChOS, a short ChOS, was obtained from Genis (Siglufj€ orður, Iceland). It has been characterized7,36 to be 60% deacetylated with 78.5% of the material having a degree of polymerization (DP) 2-6. Chitosan 75% deacetylated chitosan (Cat. no. C3646, Sigma-Aldrich, St. Luis, MO) was dissolved in 1M NaOH solution and kept at 90 C for 1 h to destroy endotoxins.30 Centrifuged, washed in ddH2O and five times in PBS. Aliquoted and kept at 4 C. ChOS lactate (Cat. no. 523682, Sigma-Aldrich) was dissolved in endotoxin free PBS, vortexed, and sterile filtered (0.22 lm). The material has been characterized by the manufacturer as 90% deacetylated with average Mn 5,000, which corresponds to DP 18. Other chemicals used were LPS (lipopolysaccharide) from E. coli 055:B5 (Cat. no. L6529) and cytochalasin D (Cat. no. PHZ1063, Sigma-Aldrich). Cytotoxicity assay Cells were seeded into a 96 well plate and treated for 24 h with test substances and cytotoxicity assessed using an XTT assay (Cat. no. 30-1011K, ATCC) following manufacturers protocol. Protein analysis Proteins in cell medium were precipitated by adding 10 volumes of medium into 40 volumes of ice cold acetone and incubated at 220 C for 1 h, followed by centrifugation at 15,000 g for 10 min. The resulting protein pellet was air dried for 20–30 min and resuspended in one volume 2x SDS loading buffer (4% SDS, 20% Glycerol, 120 mM Tris, 5% b-mercaptoethanol) and incubated at 80 C with shaking for 20 min. Whole cell lysates were extracted with RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% TritonX 100, protease inhibitors). Protein content of whole cell lysates was determined with Bradford reagent (B6916 Sigma) for equal loading. For medium equal volumes were precipitated and loaded and signal normalized with cell numbers at the end of the experiment (using crystal violet). Proteins were separated on a 12.5% SDSPAGE gel followed by transfer to a nitrocellulose membrane (Cat. no. 741290, Macherey-Nagel, D€ uren, Germany). Membranes were incubated in blocking buffer (5% milk TBS solution) for 1 h and overnight at 4 C in blocking buffer supplemented with 0.1% Tween (Sigma-Aldrich), anti-YKL40 (1:1,000, Cat. no. 4815, Quidel, San Diego, CA), anti-Chit1 (1:1,000, Cat. no. HPA010575, Sigma-Aldrich), anti-a-actin (1:10,000, Cat. no. MAB1501, Millipore, Darmstadt, Germany), or anti-Caspase-1 (1:500, Cat. no. ab17820, Abcam, Cambridge, England). The membrane was washed with TBST (TBS, 0.1% Tween) and incubated with fluorescent secondary antibody (1:20,000, Cat. no. 926-32213 & 92668072, LI-COR, Lincoln, NE) for 1 h at room temperature. After incubation, the membrane was washed in TBST, scanned in the Odyssey imaging system and signal quantified using the Image Studio 2.0 software (LI-COR). ELISA for
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FIGURE 1. ChOS lactate decreases YKL-40 and Chit-1 secretion of THP-1 macrophages. (a) YKL-40, (b) Chit-1, (c) TNF-a, and (d) IL-1b secretion in THP-1 macrophages after 24 h stimulation with 100 mg/mL ChOS lactate, chitosan, TChOS, 100 ng/mL LPS, or untreated (medium). Error bars are standard error (N 5 6, *p < 0.05; **p < 0.01; ***p < 0.001).
TNF-a and IL-1b (DuoSet ELISA, Cat. no. DY210 & DY201, R&D Systems, Minneapolis, MN) on supernatant from cells stimulated with the test substances was performed according to manufacturer instructions. Data processing and statistical analysis Western blot and ELISA values from primary macrophages were normalized with crystal violet optical density values, to adjust for cell numbers. Secretion index of Chit-1 and YKL-40 was obtained by normalizing western blot signals to cell numbers and calculating the relative change in secretion compared to untreated replicates, this was performed independently for every donor. Statistical analysis was performed using the R statistical environment. To evaluate the effect of different chitosan derivatives, one-way ANOVA with Tukey’s Multiple Comparison Post-Test was used. For the analysis of the toxicity data and for primary cells from human donors, two-way ANOVA was used. Welch two sample t test was used; p < 0.05 was considered statistically significant.
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RESULTS
Experiments were initially performed using a human monocyte cell line (THP-1). These cells can be differentiated toward a macrophage phenotype35 and are frequently used as a model of human macrophages. Chitosan, and two chitosan derivatives—a large ChOS with high levels of deactylation—ChOS lactate and a small ChOS, with low levels of deacetylation—TChOS, were added to differentiated THP-1 cells and levels of YKL-40 and Chit-1 in the medium were quantitated with western analysis 24 h later [Fig. 1(A,B)]. Samples were also analyzed with ELISA, which showed that around 100 ng/mL of YKL-40 were present in the medium after 24 h (data not shown). Comparing both methods of protein quantification directly showed that the comparative quantification from western blots was identical to the quantitative data from the ELISA. Chitinase assay was not performed, as the chemicals under study can inhibit chitinase activity,7,8 and activity is therefore not a good indicator of protein levels. As can be seen in Figure 1(A), only ChOS lactate lead to a marked reduction of YKL-40 and Chit-1 in
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FIGURE 2. ChOS lactate is cytotoxic and activates caspase 1. (a) XTT assay of THP-1 macrophages after 24 h stimulation with 100 mg/mL ChOS lactate, chitosan, TChOS, untreated (medium; N 5 4). (C) concentration gradient of ChOS lactate from 10–120 mg/mL. Error bars are standard error (N 5 3, *p < 0.05; **p < 0.01). (c) IL-1b secretion from THP-1 macrophages primed with 100 ng/mL LPS and with and without inhibiting phagocytosis with 1 mg/mL cytochalasin D prior to stimulation with 100 mg/mL ChOS lactate or chitosan for 24 h. (d) Cleavage of caspase 1 (p45) to active caspase 1 (p20) in THP-1 macrophages after LPS priming (100 ng/mL) and ChOS lactate (100 mg/mL) stimulation for 1, 2, 3, or 4 h.
medium secreted by differentiated THP-1 cells. As YKL-40 levels have been shown to regulate inflammasome activation in the mouse,24 IL-1b secretion was also measured. This showed that lowering of YKL-40 levels was concomitant with an increase in IL-1b secretion, suggesting that ChOS lactate might lead to inflammasome activation [Figs. 1(D) and 2(D)]. No increase in the secretion of TNF-a, another marker of proinflammatory response, was detected [Fig. 1(C)]. This makes proinflammatory contamination, such as the presence of endotoxins, unlikely.36–38 As seen in Figure 1(D), chitosan also led to an increase in IL-1b secretion characteristic of inflammasome activation, as previously published.30 Lowering of YKL-40 secretion by the THP-1 cells could be due to other factors than direct effect of ChOS lactate and cytotoxicity analysis showed that levels of ChOS lactate above 40 lg/ mL were cytotoxic [Fig. 2(A,B)]. The other chemicals tested did not show cytotoxicity. No reduction in YKL-40 and Chit-1 was observed in THP-1 cells at 40 lg/mL ChOS lactate (data not shown). Hence the lowering of YKL-40 in THP-1 cells above the concentrations of 40 lg/mL is at least in part due
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to the toxic effects of ChOS lactate. As seen in Figure 1(D), ChOS lactate caused increased secretion of IL-1b, indicative of inflammasome activation. This effect was reduced when phagocytosis was inhibited [Fig. 2(C)], showing that phagocytosis is pivotal for this effect, as has previously been shown.30 The main objective of this study was to examine the response of primary cells to chitosan and its derivatives, in terms of chitinase expression. Adding ChOS lactate and chitosan to primary human macrophages also led to a reduction of secreted YKL-40, but no difference in Chit-1 secretion was detected (Fig. 3). In fact, as no changes were detected in Chit-1 secretion by primary cells, it can be considered as secretion control for YKL-40. The activation of the inflammasome, is a two-step process and can be affected by first adding the bacterial endotoxin LPS. However, treating the cells with LPS prior to chitosan treatment gave similar results even though ChOS lactate concentration was decreased to 20 mg/mL [Fig. 3(C,D)]. ChOS lactate and chitosan had a toxic effect on the primary macrophages [Fig. 4(A)], but only ChOS lactate led to
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FIGURE 3. ChOS lactate and chitosan decrease YKL-40 secretion in primary macrophages. (a) YKL-40 and (b) Chit-1 secretion in primary macrophages after 24 h stimulation with 100 mg/mL ChOS lactate, chitosan, TChOS, or untreated (medium) (5 separate donors). (c) YKL-40 and (d) Chit-1 secretion in primary macrophages after 100 ng/mL LPS priming and 24 h stimulation with 20 mg/mL ChOS lactate or 100 mg/mL chitosan, TChOS or untreated (medium; 3 separate donors). (*p < 0.05; **p < 0.01; ***p < 0.001).
an increase in IL-1b secretion at subtoxic levels [Fig. 4(D)]. Slight increase in secretion of IL-1b can be observed, with added chitosan [Fig. 4(D)]. An increase in TNF-a was detected in the primary cells for ChOS lactate [Fig. 4(C)]. TChOS, which has lower level of deacetylation, and lower DP value showed no effect on the secretion of the chitinases examined and no effect on activation of the inflammasome or expression of TNF-a in either cell type.
DISCUSSION
Various chitin and chitosan derivatives are used as biomaterials, and can be present during infections. Shorter derivates (ChOS) are generated by chitinase activity in vivo, and do potentially have different bioactivity. It is therefore impor-
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tant to understand how chitin and chitosan affect the endogenous chitinases. Chitinases can also affect inflammation directly.24 This study shows that chitosan and chitosan derivatives lead to a reduction in the secretion of the inactive chitinase YKL-40 by primary human macrophages. This correlates with an increase in inflammasome activation, especially for larger highly deacetylated ChOS species. Further studies will determine whether this is a direct causal effect as in the mouse.24 No changes in the levels of the active chitinase Chit-1 were observed for any of the materials tested on primary macrophages. Hence, it is unlikely that chitosan or chitosan derivatives positively affect Chit-1 secretion in human macrophages, as has been previously suggested.27 A ChOS with high levels of deacetylation-ChOS lactate, which has been suggested as a suitable transfection
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FIGURE 4. ChOS lactate is cytotoxic and activates the inflammasome in primary macrophages. (a) XTT assay of primary macrophages after 24 h stimulation with 100 mg/mL ChOS lactate, chitosan, TChOS, untreated (medium; 3 separate donors) or (b) concentration gradient of ChOS lactate from 20 to 100 mg/mL, without (4 separate donors) and with (2 separate donors) 1 mg/mL cytochalasin D phagocytosis inhibition. (c) TNF-a and (d) IL-1b secretion in primary macrophages after 100 ng/mL LPS priming and 24 h stimulation with 20 mg/mL ChOS lactate or 100 mg/mL chitosan, TChOS or untreated (medium; 3 separate donors). Error bars are standard errors (*p < 0.05; ***p < 0.001).
agent39 and is bactericidal,40 had a toxic effect on both THP-1 macrophages and primary macrophages at high concentrations. ChOS lactate should therefore not be used experimentally, without careful analysis of its harmful effects on macrophages and possibly other cell types. Our data also shows that toxic effect of chitosan derivatives could lead to reduction of YKL-40 for example, in vivo. Two types of human macrophages were used in this study, a cell line and primary cells. They showed different responses to the chemicals under study. As expected with the primary cells, there was larger variation that is, donor variation, when compared with results from the cell line. Chitosan led to a reduction of YKL-40 in primary human macrophages [Fig. 3(A,C)], no reduction was seen in the macrophage cell line [Fig. 1(A)]. In addition, at subtoxic levels ChOS lactate did not affect YKL-40 secretion of THP-1
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cells. THP-1 cells therefore do not recapitulate fully the cellular response to chitosan, which might make them unsuitable for studies of the bioactivity of chitosan, at least with regards to inflammation. It is likely that the differences in response are either due to cell line specific changes, amount of serum present in the cultures or partial activation of the cell line macrophages, which can occur during differentiation.41 Further studies of differentially activated primary macrophages (classically and alternatively) would establish this. The focus of this study was the response of primary macrophages, more representative of the in vivo settings. Primary cells showed reduced secretion of YKL-40 when treated with both chitosan and large highly deacetylated ChOS. Only the large ChOS led to inflammasome activation, as determined by IL-1b secretion. Priming the cells with
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LPS, did not change the effect on the reduction of YKL-40. There was a clear correlation between lower levels of YKL40 and higher levels of IL-1b. Hence, chitosan, and more so, large highly deacetylated ChOS, lead to reduced secretion of YKL-40 and concomitantly more IL-1b was secreted. Chit-1 levels were not affected. Our data therefore supports the model of YKL-40 acting as a suppressor of inflammasome activation. The smaller ChOS (TChOS) did not have any detectable effect on the expression of the active chitinase Chit-1 or the inactive chitinase YKL-40 from human macrophages, neither directly or when the cells were primed with LPS previously to assess activation of the inflammasome. Also, TChOS did not affect secretion of TNF-a. Both the cell line and the primary cells gave the same results, in this regard, and in this study this compound is neither proinflammatory nor affects secretion of YKL-40 and Chit-1. This is in agreement with previous studies, that chitin and its close derivatives are usually not very active when it comes to immune response30 or possibly due to the size of TChOS. It has been reported that chitosan can lead to reduction of angiogenesis42,43 and affects the regulation of inflammation.30 The chitosans tested in this study led to a clear reduction of YKL-40 secretion in primary macrophages and could subsequently result in a reduction of angiogenesis and an increase in inflammation via inflammasome dysregulation. In cases where reduced angiogenesis or higher levels of inflammation are a concern, chitosan and especially shorter chitosan species should be avoided. Chitosan and its close derivatives negatively affect YKL-40 secretion and positively affect inflammasome activation in primary human macrophages, while smaller ChOS, similar to chitin with regards to deacetylation, do not. The former are therefore suitable for studies of infection and inflammation in primary macrophages, while the latter show more promise as inert biomaterials and do not affect YKL-40 secretion in human macrophages. Chitosan can be proinflammatory, this study shows that part of the mechanisms behind this effect can be via the reduction of the secretion of the anti-inflammatory YKL-40. ACKNOWLEDGMENTS
The authors would also like to thank Dr. Leifur Thorsteinsson for assisting with isolation of primary cells and Genis Inc. for providing material and other assistance. REFERENCES 1. Rinaudo M. Chitin and chitosan: Properties and applications. Prog Polym Sci 2006;31:603–632. 2. Pillai CKS, Paul W, Sharma CP. Chitin and chitosan polymers: Chemistry, solubility and fiber formation. Prog Polym Sci 2009;34: 641–678. 3. Kumar MN, Muzzarelli RA, Muzzarelli C, Sashiwa H, Domb AJ. Chitosan chemistry and pharmaceutical perspectives. Chem Rev 2004;104:6017–6084. 4. Jayakumar R, Prabaharan M, Nair SV, Tamura H. Novel chitin and chitosan nanofibers in biomedical applications. Biotechnol Adv 2010;28:142–150. 5. Aam BB, Heggset EB, Norberg AL, Sorlie M, Varum KM, Eijsink VG. Production of chitooligosaccharides and their potential applications in medicine. Marine Drugs 2010;8:1482–1517.
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monocytic leukemia cell line (THP-1). Int J Cancer 1980;26:171– 176. Daigneault M, Preston JA, Marriott HM, Whyte MK, Dockrell DH. The identification of markers of macrophage differentiation in PMA-stimulated THP-1 cells and monocyte-derived macrophages. PLoS One 2010;5:e8668. Lieder R, Gaware VS, Thormodsson F, Einarsson JM, Ng CH, Gislason J, Masson M, Petersen PH, Sigurjonsson OE. Endotoxins affect bioactivity of chitosan derivatives in cultures of bone marrow-derived human mesenchymal stem cells. Acta Biomater 2013;9:4771–4778. Lieder R, Petersen PH, Sigurjonsson OE. Endotoxins-the Invisible Companion in Biomaterials Research. Tissue Eng Part B Rev 2013;19:391–402. Lieder R, Gaware VG, Thromodsson F, Masson M, Peterssen PH, Sigurjonsson OE. Endotoxins - the real reason for the bioactivity in your in vitro cell assays? J Tissue Eng Regen Med 2012;6:356–356. Weecharangsan W, Opanasopit P, Ngawhirunpat T, Apirakaramwong A, Rojanarata T, Ruktanonchai U, Lee RJ. Evaluation of chitosan salts as non-viral gene vectors in CHO-K1 cells. Int J Pharm 2008;348:161–168. Rabea EI, Badawy MET, Stevens CV, Smagghe G, Steurbaut W. Chitosan as antimicrobial agent: Applications and mode of action. Biomacromolecules 2003;4:1457–1465. Maess MB, Wittig B, Cignarella A, Lorkowski S. Reduced PMA enhances the responsiveness of transfected THP-1 macrophages to polarizing stimuli. J Immunol Methods 2014;402:76–81. Mathieu C, Chevrier A, Lascau-Coman V, Rivard GE, Hoemann CD. Stereological analysis of subchondral angiogenesis induced by chitosan and coagulation factors in microdrilled articular cartilage defects. Osteoarthritis Cartilage 2013;21:849–859. Wu H, Aam BB, Wang W, Norberg AL, Sørlie M, Eijsink VGH, Du Y. Inhibition of angiogenesis by chitooligosaccharides with specific degrees of acetylation and polymerization. Carbohydr Polym 2012;89:511–518.
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Faculty of Medicine, School of Health Sciences, Department of Anatomy, Biomedical Center, University of Iceland, Reykjavik 101, Iceland 2 The Blood Bank, Landspitali University Hospital, Snorrabraut 60, Reykjavik 105, Iceland 3 School of Science and Engineering, Reykjavik University, Menntavegur 1, Reykjavik 101, Iceland 4 Biomedical Center, University of Iceland, Reykjavik 101, Iceland Received 23 September 2014; revised 18 December 2014; accepted 5 February 2015 Published online 27 February 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35417 Abstract: Chitosan, the deacetylated derivative of chitin, is used as biomaterial in diverse settings. It is also found on pathogens and can be proinflammatory. Shorter derivatives of chitosan can be generated chemically or enzymatically, chitosan oligosaccharides (ChOS). There is variation in the chemical composition of ChOS, including size distribution, but in general, they have been described as inert or antiinflammatory. Active human chitinases can cleave chitin and chitosan, while inactive chitinases bind both but do not cleave. Both active and inactive chitinases have important roles in the immune response. The inactive chitinase YKL-40 is expressed highly during inflammation and has been proposed as a marker of poor prognosis. YKL-40 acts as a negative regulator of the inflammasome and as a positive regulator of angiogenesis. Levels of YKL-40 can therefore reg-
ulate levels of inflammation, the extent of angiogenesis, and the process of inflammation resolution. This study shows that chitosan leads to reduced secretion of YKL-40 by primary human macrophages and that this is concomitant with inflammasome activation. This was most pronounced with a highly deacetylated ChOS. No effect on the secretion of the active chitinase Chit-1 was detected. Smaller and more acetylated ChOS did not affect YKL-40 levels nor inflammasome activation. We conclude that this effect on the levels of YKL40 is a part of the proinflammatory mechanisms of chitosan C 2015 Wiley Periodicals, Inc. J Biomed Mater and its derivatives. V Res Part A: 103A: 2778–2785, 2015.
Key Words: chitin, chitosan, inflammation, chitinase, YKL-40
How to cite this article: Gudmundsdottir S, Lieder R, Sigurjonsson OE, Petersen PH. 2015. Chitosan leads to downregulation of YKL-40 and inflammasome activation in human macrophages. J Biomed Mater Res Part A 2015:103A:2778–2785.
INTRODUCTION
The polysaccharide chitin, the polymer of Nacetylglucosamine, is found widely in nature, including as a part of various human pathogens. Deacetylation of chitin produces chitosan, which has increased water-solubility under acidic conditions.1,2 Chemically modified chitosan in various formulations shows potential as biomaterial, drug carrier, wound dressing, and transfection agent.3,4 Chitosan can be further cleaved enzymatically or chemically to generate smaller chitosan oligosaccharides (ChOS), which show increased solubility. ChOS, which are of variable length and different degrees of deacetylation, are more resistant to further degradation.5 ChOS are able to bind to chitin binding proteins, and can act as inhibitors of chitinases.6–8 While mammalian tissues do not contain chitin or chitosan, mammalian cells can express both active and inactive extracellular chitinases, the latter being able to bind, but not cleave chitin.9,10 Both take an active part in the immune
response,11,12 and likely play a special role in the response against pathogens which contain chitin or chitosan.13–15 Active chitinases, such as Chitotriosidase-1 (Chit-1) cleave chitin and chitin derivatives and can inhibit fungal growth.16 Inactive chitinases, of which YKL-40 (also known as CHI3L1 and BRP-39) is best characterized,17 also play a role in tissue repair, tissue remodeling11,18 and angiogenesis.19 Chit-1 and YKL-40, which are the focus of this study, are secreted by macrophages, and are highly upregulated in monocyte to macrophage differentiation.20,21 YKL-40 is upregulated in a large variety of pathological conditions and can be a marker of poor prognosis.22,23 Extracellular YKL-40 has been shown to play a major role in inhibiting the inflammasome, which leads to a reduction in the secretion of the proinflammatory cytokine IL-1b.24 YKL-40 could therefore play an important role in regulation and resolution of inflammation during infection, possibly in concert with changes in activation pattern of macrophages, that is, from classically activated
Correspondence to: P. H. Petersen; e-mail: [email protected] 1354-845-5346 Contract grant sponsor: Landspitali University Hospital Research Fund Contract grant sponsor: Icelandic Student Innovation Fund; contract grant number: 1323410091
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macrophages expressing YKL-40, to alternatively activated macrophages with reduced YKL-40 expression.25 However, little is known about the regulation of YKL-40. As chitosan is present on important human pathogens26 and its cleavage by Chit-1 leads to formation of ChOS, it is important to establish whether chitosan and ChOS have a direct effect on the expression of chitinases, both active and inactive. It has been suggested that ChOS, derived from degradation of chitosan in vivo, induces the expression of Chit-1 in THP-1 cells,27 which could be considered as positive feedback. Chitosan is used in diverse settings as biomaterial and has immunomodulating effects, that is, can act either antiinflammatory28,29 or proinflammatory,30 depending on the chemical composition of the chitosan and the experimental settings. This is further complicated by the in vivo degradation of chitosan into ChOS. ChOS also have diverse potential as biomaterials, such as scaffolds in tissue engineering, pharmaceuticals, vectors for gene delivery, in wound healing and as antifungal agents,3,5 and have been shown in general, to act as anti-inflammatory agents.31–33 The goal of this study was to test in primary human macrophages, one of the principal cell type of the immune response, whether chitosan and two different ChOS species had an effect on the secretion of Chit-1 and YKL-40 and whether they affected the activation of the inflammasome.
MATERIALS AND METHODS
Cell culture The THP-1 cell line34 was obtained from ATCC (Manassas, Virginia) and cultured in RPMI 1640 (Cat. no. 52400–025) supplemented with 10% FBS (Cat. no. 10270-106), 100 U/ mL Penicillin and 100 mg/mL Streptomycin (Cat. no. 15140122, Gibco, Carlsbad, CA). THP-1 cells (5 3 105 cells/mL) were differentiated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA, Cat. no. 524400, Calbiochem, San Diego, CA) in RPMI 1640 supplemented with 2% FBS for 2 days, followed by 2 days in PMA free media.35 Primary monocytes were isolated from buffy coat of healthy human volunteers with Ficoll-PaqueTM (Cat. no. 171440-02) density gradient centrifugation and MACS CD141 magnetic beads (Cat. no. 130-050-201, Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were seeded at 7.5 3 105 monocytes/mL in RPMI 1640 Glutamax (Cat. no. 61870, Gibco) medium containing 10% pooled normal human serum and 100 U/mL and 100 mg/mL Pen/Strep respectively (Cat. no. 10378016, Gibco). After 3 days, half of the medium was exchanged for fresh medium. On day 6, cells were washed with PBS (Cat. no. 10010-056, Gibco) and fresh medium added. Cells were stimulated with test substances in serum free medium on day 9; 24 h after stimulation the medium was collected for western blot and ELISA analysis and the cells were stained with crystal violet solution. Cells were fixed with 4% formaldehyde in PBS (Cat. no. 252549, Sigma-Aldrich), washed with PBS and stained with crystal violet solution (PBS w/10% v/v ethanol, 0.1% w/v crystal violet) for 20 min. Then, cells were washed three times with dH2O and relative cell numbers deter-
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mined by dissolving the dye with 30% acetic acid and measuring optical density at 570 nm. CHEMICALS
TChOS, a short ChOS, was obtained from Genis (Siglufj€ orður, Iceland). It has been characterized7,36 to be 60% deacetylated with 78.5% of the material having a degree of polymerization (DP) 2-6. Chitosan 75% deacetylated chitosan (Cat. no. C3646, Sigma-Aldrich, St. Luis, MO) was dissolved in 1M NaOH solution and kept at 90 C for 1 h to destroy endotoxins.30 Centrifuged, washed in ddH2O and five times in PBS. Aliquoted and kept at 4 C. ChOS lactate (Cat. no. 523682, Sigma-Aldrich) was dissolved in endotoxin free PBS, vortexed, and sterile filtered (0.22 lm). The material has been characterized by the manufacturer as 90% deacetylated with average Mn 5,000, which corresponds to DP 18. Other chemicals used were LPS (lipopolysaccharide) from E. coli 055:B5 (Cat. no. L6529) and cytochalasin D (Cat. no. PHZ1063, Sigma-Aldrich). Cytotoxicity assay Cells were seeded into a 96 well plate and treated for 24 h with test substances and cytotoxicity assessed using an XTT assay (Cat. no. 30-1011K, ATCC) following manufacturers protocol. Protein analysis Proteins in cell medium were precipitated by adding 10 volumes of medium into 40 volumes of ice cold acetone and incubated at 220 C for 1 h, followed by centrifugation at 15,000 g for 10 min. The resulting protein pellet was air dried for 20–30 min and resuspended in one volume 2x SDS loading buffer (4% SDS, 20% Glycerol, 120 mM Tris, 5% b-mercaptoethanol) and incubated at 80 C with shaking for 20 min. Whole cell lysates were extracted with RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% TritonX 100, protease inhibitors). Protein content of whole cell lysates was determined with Bradford reagent (B6916 Sigma) for equal loading. For medium equal volumes were precipitated and loaded and signal normalized with cell numbers at the end of the experiment (using crystal violet). Proteins were separated on a 12.5% SDSPAGE gel followed by transfer to a nitrocellulose membrane (Cat. no. 741290, Macherey-Nagel, D€ uren, Germany). Membranes were incubated in blocking buffer (5% milk TBS solution) for 1 h and overnight at 4 C in blocking buffer supplemented with 0.1% Tween (Sigma-Aldrich), anti-YKL40 (1:1,000, Cat. no. 4815, Quidel, San Diego, CA), anti-Chit1 (1:1,000, Cat. no. HPA010575, Sigma-Aldrich), anti-a-actin (1:10,000, Cat. no. MAB1501, Millipore, Darmstadt, Germany), or anti-Caspase-1 (1:500, Cat. no. ab17820, Abcam, Cambridge, England). The membrane was washed with TBST (TBS, 0.1% Tween) and incubated with fluorescent secondary antibody (1:20,000, Cat. no. 926-32213 & 92668072, LI-COR, Lincoln, NE) for 1 h at room temperature. After incubation, the membrane was washed in TBST, scanned in the Odyssey imaging system and signal quantified using the Image Studio 2.0 software (LI-COR). ELISA for
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FIGURE 1. ChOS lactate decreases YKL-40 and Chit-1 secretion of THP-1 macrophages. (a) YKL-40, (b) Chit-1, (c) TNF-a, and (d) IL-1b secretion in THP-1 macrophages after 24 h stimulation with 100 mg/mL ChOS lactate, chitosan, TChOS, 100 ng/mL LPS, or untreated (medium). Error bars are standard error (N 5 6, *p < 0.05; **p < 0.01; ***p < 0.001).
TNF-a and IL-1b (DuoSet ELISA, Cat. no. DY210 & DY201, R&D Systems, Minneapolis, MN) on supernatant from cells stimulated with the test substances was performed according to manufacturer instructions. Data processing and statistical analysis Western blot and ELISA values from primary macrophages were normalized with crystal violet optical density values, to adjust for cell numbers. Secretion index of Chit-1 and YKL-40 was obtained by normalizing western blot signals to cell numbers and calculating the relative change in secretion compared to untreated replicates, this was performed independently for every donor. Statistical analysis was performed using the R statistical environment. To evaluate the effect of different chitosan derivatives, one-way ANOVA with Tukey’s Multiple Comparison Post-Test was used. For the analysis of the toxicity data and for primary cells from human donors, two-way ANOVA was used. Welch two sample t test was used; p < 0.05 was considered statistically significant.
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RESULTS
Experiments were initially performed using a human monocyte cell line (THP-1). These cells can be differentiated toward a macrophage phenotype35 and are frequently used as a model of human macrophages. Chitosan, and two chitosan derivatives—a large ChOS with high levels of deactylation—ChOS lactate and a small ChOS, with low levels of deacetylation—TChOS, were added to differentiated THP-1 cells and levels of YKL-40 and Chit-1 in the medium were quantitated with western analysis 24 h later [Fig. 1(A,B)]. Samples were also analyzed with ELISA, which showed that around 100 ng/mL of YKL-40 were present in the medium after 24 h (data not shown). Comparing both methods of protein quantification directly showed that the comparative quantification from western blots was identical to the quantitative data from the ELISA. Chitinase assay was not performed, as the chemicals under study can inhibit chitinase activity,7,8 and activity is therefore not a good indicator of protein levels. As can be seen in Figure 1(A), only ChOS lactate lead to a marked reduction of YKL-40 and Chit-1 in
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FIGURE 2. ChOS lactate is cytotoxic and activates caspase 1. (a) XTT assay of THP-1 macrophages after 24 h stimulation with 100 mg/mL ChOS lactate, chitosan, TChOS, untreated (medium; N 5 4). (C) concentration gradient of ChOS lactate from 10–120 mg/mL. Error bars are standard error (N 5 3, *p < 0.05; **p < 0.01). (c) IL-1b secretion from THP-1 macrophages primed with 100 ng/mL LPS and with and without inhibiting phagocytosis with 1 mg/mL cytochalasin D prior to stimulation with 100 mg/mL ChOS lactate or chitosan for 24 h. (d) Cleavage of caspase 1 (p45) to active caspase 1 (p20) in THP-1 macrophages after LPS priming (100 ng/mL) and ChOS lactate (100 mg/mL) stimulation for 1, 2, 3, or 4 h.
medium secreted by differentiated THP-1 cells. As YKL-40 levels have been shown to regulate inflammasome activation in the mouse,24 IL-1b secretion was also measured. This showed that lowering of YKL-40 levels was concomitant with an increase in IL-1b secretion, suggesting that ChOS lactate might lead to inflammasome activation [Figs. 1(D) and 2(D)]. No increase in the secretion of TNF-a, another marker of proinflammatory response, was detected [Fig. 1(C)]. This makes proinflammatory contamination, such as the presence of endotoxins, unlikely.36–38 As seen in Figure 1(D), chitosan also led to an increase in IL-1b secretion characteristic of inflammasome activation, as previously published.30 Lowering of YKL-40 secretion by the THP-1 cells could be due to other factors than direct effect of ChOS lactate and cytotoxicity analysis showed that levels of ChOS lactate above 40 lg/ mL were cytotoxic [Fig. 2(A,B)]. The other chemicals tested did not show cytotoxicity. No reduction in YKL-40 and Chit-1 was observed in THP-1 cells at 40 lg/mL ChOS lactate (data not shown). Hence the lowering of YKL-40 in THP-1 cells above the concentrations of 40 lg/mL is at least in part due
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to the toxic effects of ChOS lactate. As seen in Figure 1(D), ChOS lactate caused increased secretion of IL-1b, indicative of inflammasome activation. This effect was reduced when phagocytosis was inhibited [Fig. 2(C)], showing that phagocytosis is pivotal for this effect, as has previously been shown.30 The main objective of this study was to examine the response of primary cells to chitosan and its derivatives, in terms of chitinase expression. Adding ChOS lactate and chitosan to primary human macrophages also led to a reduction of secreted YKL-40, but no difference in Chit-1 secretion was detected (Fig. 3). In fact, as no changes were detected in Chit-1 secretion by primary cells, it can be considered as secretion control for YKL-40. The activation of the inflammasome, is a two-step process and can be affected by first adding the bacterial endotoxin LPS. However, treating the cells with LPS prior to chitosan treatment gave similar results even though ChOS lactate concentration was decreased to 20 mg/mL [Fig. 3(C,D)]. ChOS lactate and chitosan had a toxic effect on the primary macrophages [Fig. 4(A)], but only ChOS lactate led to
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FIGURE 3. ChOS lactate and chitosan decrease YKL-40 secretion in primary macrophages. (a) YKL-40 and (b) Chit-1 secretion in primary macrophages after 24 h stimulation with 100 mg/mL ChOS lactate, chitosan, TChOS, or untreated (medium) (5 separate donors). (c) YKL-40 and (d) Chit-1 secretion in primary macrophages after 100 ng/mL LPS priming and 24 h stimulation with 20 mg/mL ChOS lactate or 100 mg/mL chitosan, TChOS or untreated (medium; 3 separate donors). (*p < 0.05; **p < 0.01; ***p < 0.001).
an increase in IL-1b secretion at subtoxic levels [Fig. 4(D)]. Slight increase in secretion of IL-1b can be observed, with added chitosan [Fig. 4(D)]. An increase in TNF-a was detected in the primary cells for ChOS lactate [Fig. 4(C)]. TChOS, which has lower level of deacetylation, and lower DP value showed no effect on the secretion of the chitinases examined and no effect on activation of the inflammasome or expression of TNF-a in either cell type.
DISCUSSION
Various chitin and chitosan derivatives are used as biomaterials, and can be present during infections. Shorter derivates (ChOS) are generated by chitinase activity in vivo, and do potentially have different bioactivity. It is therefore impor-
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tant to understand how chitin and chitosan affect the endogenous chitinases. Chitinases can also affect inflammation directly.24 This study shows that chitosan and chitosan derivatives lead to a reduction in the secretion of the inactive chitinase YKL-40 by primary human macrophages. This correlates with an increase in inflammasome activation, especially for larger highly deacetylated ChOS species. Further studies will determine whether this is a direct causal effect as in the mouse.24 No changes in the levels of the active chitinase Chit-1 were observed for any of the materials tested on primary macrophages. Hence, it is unlikely that chitosan or chitosan derivatives positively affect Chit-1 secretion in human macrophages, as has been previously suggested.27 A ChOS with high levels of deacetylation-ChOS lactate, which has been suggested as a suitable transfection
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FIGURE 4. ChOS lactate is cytotoxic and activates the inflammasome in primary macrophages. (a) XTT assay of primary macrophages after 24 h stimulation with 100 mg/mL ChOS lactate, chitosan, TChOS, untreated (medium; 3 separate donors) or (b) concentration gradient of ChOS lactate from 20 to 100 mg/mL, without (4 separate donors) and with (2 separate donors) 1 mg/mL cytochalasin D phagocytosis inhibition. (c) TNF-a and (d) IL-1b secretion in primary macrophages after 100 ng/mL LPS priming and 24 h stimulation with 20 mg/mL ChOS lactate or 100 mg/mL chitosan, TChOS or untreated (medium; 3 separate donors). Error bars are standard errors (*p < 0.05; ***p < 0.001).
agent39 and is bactericidal,40 had a toxic effect on both THP-1 macrophages and primary macrophages at high concentrations. ChOS lactate should therefore not be used experimentally, without careful analysis of its harmful effects on macrophages and possibly other cell types. Our data also shows that toxic effect of chitosan derivatives could lead to reduction of YKL-40 for example, in vivo. Two types of human macrophages were used in this study, a cell line and primary cells. They showed different responses to the chemicals under study. As expected with the primary cells, there was larger variation that is, donor variation, when compared with results from the cell line. Chitosan led to a reduction of YKL-40 in primary human macrophages [Fig. 3(A,C)], no reduction was seen in the macrophage cell line [Fig. 1(A)]. In addition, at subtoxic levels ChOS lactate did not affect YKL-40 secretion of THP-1
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cells. THP-1 cells therefore do not recapitulate fully the cellular response to chitosan, which might make them unsuitable for studies of the bioactivity of chitosan, at least with regards to inflammation. It is likely that the differences in response are either due to cell line specific changes, amount of serum present in the cultures or partial activation of the cell line macrophages, which can occur during differentiation.41 Further studies of differentially activated primary macrophages (classically and alternatively) would establish this. The focus of this study was the response of primary macrophages, more representative of the in vivo settings. Primary cells showed reduced secretion of YKL-40 when treated with both chitosan and large highly deacetylated ChOS. Only the large ChOS led to inflammasome activation, as determined by IL-1b secretion. Priming the cells with
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LPS, did not change the effect on the reduction of YKL-40. There was a clear correlation between lower levels of YKL40 and higher levels of IL-1b. Hence, chitosan, and more so, large highly deacetylated ChOS, lead to reduced secretion of YKL-40 and concomitantly more IL-1b was secreted. Chit-1 levels were not affected. Our data therefore supports the model of YKL-40 acting as a suppressor of inflammasome activation. The smaller ChOS (TChOS) did not have any detectable effect on the expression of the active chitinase Chit-1 or the inactive chitinase YKL-40 from human macrophages, neither directly or when the cells were primed with LPS previously to assess activation of the inflammasome. Also, TChOS did not affect secretion of TNF-a. Both the cell line and the primary cells gave the same results, in this regard, and in this study this compound is neither proinflammatory nor affects secretion of YKL-40 and Chit-1. This is in agreement with previous studies, that chitin and its close derivatives are usually not very active when it comes to immune response30 or possibly due to the size of TChOS. It has been reported that chitosan can lead to reduction of angiogenesis42,43 and affects the regulation of inflammation.30 The chitosans tested in this study led to a clear reduction of YKL-40 secretion in primary macrophages and could subsequently result in a reduction of angiogenesis and an increase in inflammation via inflammasome dysregulation. In cases where reduced angiogenesis or higher levels of inflammation are a concern, chitosan and especially shorter chitosan species should be avoided. Chitosan and its close derivatives negatively affect YKL-40 secretion and positively affect inflammasome activation in primary human macrophages, while smaller ChOS, similar to chitin with regards to deacetylation, do not. The former are therefore suitable for studies of infection and inflammation in primary macrophages, while the latter show more promise as inert biomaterials and do not affect YKL-40 secretion in human macrophages. Chitosan can be proinflammatory, this study shows that part of the mechanisms behind this effect can be via the reduction of the secretion of the anti-inflammatory YKL-40. ACKNOWLEDGMENTS
The authors would also like to thank Dr. Leifur Thorsteinsson for assisting with isolation of primary cells and Genis Inc. for providing material and other assistance. REFERENCES 1. Rinaudo M. Chitin and chitosan: Properties and applications. Prog Polym Sci 2006;31:603–632. 2. Pillai CKS, Paul W, Sharma CP. Chitin and chitosan polymers: Chemistry, solubility and fiber formation. Prog Polym Sci 2009;34: 641–678. 3. Kumar MN, Muzzarelli RA, Muzzarelli C, Sashiwa H, Domb AJ. Chitosan chemistry and pharmaceutical perspectives. Chem Rev 2004;104:6017–6084. 4. Jayakumar R, Prabaharan M, Nair SV, Tamura H. Novel chitin and chitosan nanofibers in biomedical applications. Biotechnol Adv 2010;28:142–150. 5. Aam BB, Heggset EB, Norberg AL, Sorlie M, Varum KM, Eijsink VG. Production of chitooligosaccharides and their potential applications in medicine. Marine Drugs 2010;8:1482–1517.
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6. Cederkvist FH, Parmer MP, Varum KM, Eijsink VGH, Sorlie M. Inhibition of a family 18 chitinase by chitooligosaccharides. Carbohydr Polym 2008;74:41–49. 7. Einarsson JM, Bahrke S, Sigurdsson BT, Ng CH, Petersen PH, Sigurjonsson OE, Jonsson JH, Gislason J, Thormodsson FR, Peter MG. Partially acetylated chitooligosaccharides bind to YKL40 and stimulate growth of human osteoarthritic chondrocytes. Biochem Biophys Res Commun 2013;434:298–304. 8. Chen L, Zhou Y, Qu MB, Zhao Y, Yang Q. Fully deacetylated chitooligosaccharides act as efficient glycoside hydrolase family 18 chitinase inhibitors. J Biol Chem 2014;289:17932–17940. 9. Fusetti F, Pijning T, Kalk KH, Bos E, Dijkstra BW. Crystal structure and carbohydrate-binding properties of the human cartilage glycoprotein-39. J Biol Chem 2003;278:37753–37760. 10. Schimpl M, Rush CL, Betou M, Eggleston IM, Recklies AD, van Aalten DMF. Human YKL-39 is a pseudo-chitinase with retained chitooligosaccharide-binding properties. Biochem J 2012;446:149– 157. 11. Lee CG, Da Silva CA, Dela Cruz CS, Ahangari F, Ma B, Kang MJ, He CH, Takyar S, Elias JA. Role of chitin and chitinase/chitinaselike proteins in inflammation, tissue remodeling, and injury. Annu Rev Physiol 2011;73:479–501. 12. Lee CG, Hartl D, Lee GR, Koller B, Matsuura H, Da Silva CA, Sohn MH, Cohn L, Homer RJ, Kozhich AA, Humbles A, Kearley J, Coyle A, Chupp G, Reed J, Flavell RA, Elias JA. Role of breast regression protein 39 (BRP-39)/chitinase 3-like-1 in Th2 and IL-13induced tissue responses and apoptosis. J Exp Med 2009;206: 1149–1166. 13. Labadaridis I, Dimitriou E, Theodorakis M, Kafalidis G, Velegraki A, Michelakakis H. Chitotriosidase in neonates with fungal and bacterial infections. Arch Dis Child Fetal Neonatal Ed 2005;90: F531–F532. 14. Barone R, Simpore` J, Malaguarnera L, Pignatelli S, Musumeci S. Plasma chitotriosidase activity in acute Plasmodium falciparum malaria. J Trop Pediatr 2003;49:63–64. 15. Goldman DL, Li X, Tsirilakis K, Andrade C, Casadevall A, Vicencio AG. Increased chitinase expression and fungal-specific antibodies in the bronchoalveolar lavage fluid of asthmatic children. Clin Exp Allergy 2012;42:523–530. 16. van Eijk M, van Roomen CPAA, Renkema GH, Bussink AP, Andrews L, Blommaart EFC, Sugar A, Verhoeven AJ, Boot RG, Aerts JMFG. Characterization of human phagocyte-derived chitotriosidase, a component of innate immunity. Int Immunol 2005; 17:1505–1512. 17. Prakash M, Bodas M, Prakash D, Nawani N, Khetmalas M, Mandal A, Eriksson C. Diverse pathological implications of YKL-40: Answers may lie in ’outside-in’ signaling. Cell Signal 2013;25:1567–1573. 18. Recklies AD, White C, Ling H. The chitinase 3-like protein human cartilage glycoprotein 39 (HC-gp39) stimulates proliferation of human connective-tissue cells and activates both extracellular signal-regulated kinase- and protein kinase B-mediated signalling pathways. Biochem J 2002;365(Pt 1):119–126. 19. Shao R, Hamel K, Petersen L, Cao QJ, Arenas RB, Bigelow C, Bentley B, Yan W. YKL-40, a secreted glycoprotein, promotes tumor angiogenesis. Oncogene 2009;28:4456–4468. 20. Rehli M, Niller HH, Ammon C, Langmann S, Schwarzfischer L, Andreesen R, Krause SW. Transcriptional regulation of CHI3L1, a marker gene for late stages of macrophage differentiation. J Biol Chem 2003;278:44058–44067. 21. Di Rosa M, Malaguarnera G, De Gregorio C, Drago F, Malaguarnera L. Evaluation of CHI3L-1 and CHIT-1 expression in differentiated and polarized macrophages. Inflammation 2013;36:482–492. 22. Johansen JS, Cintin C, Jorgensen M, Kamby C, Price PA. Serum YKL-40: A new potential marker of prognosis and location of metastases of patients with recurrent breast cancer. Eur J Cancer 1995;31A:1437–1442. 23. Schultz NA, Johansen JS. YKL-40-A protein in the field of translational medicine: A role as a biomarker in cancer patients? Cancers (Basel) 2010;2:1453–1491. 24. Dela Cruz CS, Liu W, He CH, Jacoby A, Gornitzky A, Ma B, Flavell R, Lee CG, Elias JA. Chitinase 3-like-1 promotes Streptococcus pneumoniae killing and augments host tolerance to lung antibacterial responses. Cell Host Microbe 2012;12:34–46.
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