A humanized Anti-YKL-40 antibody inhibits tumor development.

Drugs specifically targeting YKL-40, an over-expressed gene (CHI3L1) in various diseases remain developed. The current study is to create a humanized anti-YKL-40 neutralizing antibody and characterize its potentially therapeutic signature. We utilized in silico CDR-grafting bioinformatics to replace the complementarity determining regions (CDRs) of human IgG1 with mouse CDRs of our previously established anti-YKL-40 antibody (mAY). In fifteen candidates (VL1-3/VH1-5) of heavy and light chain variable region combination, one antibody L3H4 named Rosazumab demonstrated strong binding affinity with YKL-40 (KD = 4.645 × 10-8 M) and high homology with human IgG (80 %). In addition, we established different overlapping amino acid peptides of YKL-40 and found that Rosazumab specifically bound to residues K337, K342, and R344, the KR-rich functional domain of YKL-40. Rosazumab inhibited migration and tube formation of YKL-40-expressing tumor cells and induced tumor cell apoptosis. Mechanistically, Rosazumab induced interaction of N-cadherin with ß-catenin and activation of downstream MST1/RASSF1/Histone H2B axis, leading to chromosomal DNA breakage and cell apoptosis. Treatment of xenografted tumor mice with Rosazumab twice a week for 4 weeks inhibited tumor growth and angiogenesis, but induced tumor apoptosis. Rosazumab injected in mice distributed to blood, tumor, and other multiple organs, but did not impact in function or structure of liver and kidney, indicating non-detectable toxicity in vivo. Collectively, the study is the first one to demonstrate that a humanized YKL-40 neutralizing antibody offers a valuable means to block tumor development.

Fully human chitinase-3 like-1 monoclonal antibody inhibits tumor growth, fibrosis, angiogenesis, and immune cell remodeling in lung, pancreatic, and colorectal cancers.

Considering the limited efficacy of current therapies in lung, colorectal, and pancreatic cancers, innovative combination treatments with diverse mechanisms of action are needed to improve patients' outcomes. Chitinase-3 like-1 protein (CHI3L1) emerges as a versatile factor with significant implications in various diseases, particularly cancers, fostering an immunosuppressive tumor microenvironment for cancer progression. Therefore, pre-clinical validation is imperative to fully realize its potential in cancer treatment. We developed phage display-derived fully human monoclonal CHI3L1 neutralizing antibodies (nAbs) and verified the nAbs-antigen binding affinity and specificity in lung, pancreatic and colorectal cancer cell lines. Tumor growth signals, proliferation and migration ability were all reduced by CHI3L1 nAbs in vitro. Orthotopic or subcutaneous tumor mice model and humanized mouse model were established for characterizing the anti-tumor properties of two CHI3L1 nAb leads. Importantly, CHI3L1 nAbs not only inhibited tumor growth but also mitigated fibrosis, angiogenesis, and restored immunostimulatory functions of immune cells in pancreatic, lung, and colorectal tumor mice models. Mechanistically, CHI3L1 nAbs directly suppressed the activation of pancreatic stellate cells and the transformation of macrophages into myofibroblasts, thereby attenuating fibrosis. These findings strongly support the therapeutic potential of CHI3L1 nAbs in overcoming clinical challenges, including the failure of gemcitabine in pancreatic cancer.

CHI3L1 regulates PD-L1 and anti-CHI3L1-PD-1 antibody elicits synergistic antitumor responses.

Evasion of the immune response is a hallmark of cancer, and programmed cell death 1 (PD-1) and PD-1 ligand 1 (PD-L1) are major mediators of this immunosuppression. Chitinase 3-like 1 (CHI3L1) is induced in many cancers, where it portends a poor prognosis and contributes to tumor metastasis and spread. However, the mechanism(s) that CHI3L1 uses in metastasis have not been defined. Here we demonstrate that CHI3L1 regulates the expression of PD-L1, PD-L2, PD-1, LAG3, and TIM3 and plays a critical role in melanoma progression and lymphatic spread. CHI3L1 also contributed to IFN-γ-stimulated macrophage PD-L1 expression, and RIG-like helicase innate immunity suppressed CHI3L1, PD-L1, and melanoma progression. Individual antibodies against CHI3L1 or PD-1 had discrete antitumor effects and additive antitumor responses in metastasis models and T cell-tumor cell cocultures when administered simultaneously. Synergistic cytotoxic tumor cell death was seen in T cell-tumor cell cocultures, and significantly enhanced antitumor responses were seen in in vivo tumor models treated with bispecific antibodies that simultaneously target CHI3L1 and PD-1. CHI3L1 contributes to tumor progression by stimulating the PD-1/PD-L1 axis and other checkpoint molecules. The simultaneous targeting of CHI3L1 and the PD-1/PD-L1 axis with individual and, more powerfully, with bispecific antibodies represents a promising therapy for pulmonary metastasis and progression.

Chitinase 3-like-1 is a therapeutic target that mediates the effects of aging in COVID-19.

COVID-19 is caused by SARS-CoV-2 (SC2) and is more prevalent and severe in elderly and patients with comorbid diseases (CM). Because chitinase 3-like-1 (CHI3L1) is induced during aging and CM, the relationships between CHI3L1 and SC2 were investigated. Here, we demonstrate that CHI3L1 is a potent stimulator of the SC2 receptor angiotensin converting enzyme 2 (ACE2) and viral spike protein priming proteases (SPP), that ACE2 and SPP are induced during aging, and that anti-CHI3L1, kasugamycin, and inhibitors of phosphorylation abrogate these ACE2- and SPP-inductive events. Human studies also demonstrate that the levels of circulating CHI3L1 are increased in the elderly and patients with CM, where they correlate with COVID-19 severity. These studies demonstrate that CHI3L1 is a potent stimulator of ACE2 and SPP, that this induction is a major mechanism contributing to the effects of aging during SC2 infection, and that CHI3L1 co-opts the CHI3L1 axis to augment SC2 infection. CHI3L1 plays a critical role in the pathogenesis of and is an attractive therapeutic target in COVID-19.

The effects of YKL-40 on angiogenic potential of HUVECs are partly mediated by syndecan-4.

Background: YKL-40, a secreted glycoprotein, has a role in promoting tumor angiogenesis through syndecan-1 receptor. Syndecan-4 is a member of syndecan family. However, the effects of YKL-40 on migration and tube formation of human umbilical vein cells (HUVECs) mediated by syndecan-4 receptor are unknown. Materials and methods: HUVECs were transfected with lentivirus encoding syndecan-4 short hairpin (sh) RNAs (lenti-synd4 shRNAs) and the efficiency of transfection was measured using qRT-PCR and western blotting. The effects of recombinant protein of YKL-40 on migration and angiogenesis of HUVECs adjusted by syndecan-4 were determined by wound healing and tube formation assay. The expressions of protein kinase Cα (PKCα) and extracellular signal regulated kinases (ERKs) 1 and 2 (ERK1/2) in HUVECs were measured using western blotting. Results: The mRNA and protein expression of syndecan-4 were significantly decreased in HUVECs successfully transfected with lenti-synd4 shRNAs. Lenti-synd4 shRNAs remarkably inhibited the migration and tube formation of HUVECs stimulated by recombinant protein of YKL-40. The levels of PKCα and ratio of p-ERK1/2 to ERK1/2 in HUVECs were also decreased by down-regulating syndecan-4. Conclusion: The effects of YKL-40 on migration and tube formation of HUVECs are partly inhibited by knock-downing syndecan-4 through suppressing PKCα and ERK1/2 signaling pathways.

Perspective: targeting VEGF-A and YKL-40 in glioblastoma - matter matters.

Glioblastomas (GBM) are heterogeneous highly vascular brain tumors exploiting the unique microenvironment in the brain to resist treatment and anti-tumor responses. Anti-angiogenic agents, immunotherapy, and targeted therapy have been studied extensively in GBM patients over a number of decades with minimal success. Despite maximal efforts, prognosis remains dismal with an overall survival of approximately 15 months.Bevacizumab, a humanized anti-vascular endothelial growth factor (VEGF) antibody, underwent accelerated approval by the U.S. Food and Drug Administration in 2009 for the treatment of recurrent GBM based on promising preclinical and early clinical studies. Unfortunately, subsequent clinical trials did not find overall survival benefit. Pursuing pleiotropic targets and leaning toward multitarget strategies may be a key to more effective therapeutic intervention in GBM, but preclinical evaluation requires careful consideration of model choices. In this study, we discuss bevacizumab resistance, dual targeting of pro-angiogenic modulators VEGF and YKL-40 in the context of brain tumor microenvironment, and how model choice impacts study conclusions and its translational significance.

K284-6111 alleviates memory impairment and neuroinflammation in Tg2576 mice by inhibition of Chitinase-3-like 1 regulating ERK-dependent PTX3 pathway.

BACKGROUND: Alzheimer's disease (AD) is one of the most prevalent neurodegenerative disorders characterized by gradual memory loss and neuropsychiatric symptoms. We have previously demonstrated that the 2-({3-[2-(1-cyclohexene-1-yl)ethyl]-6,7-dimethoxy-4-oxo-3,4-dihydro-2-quinazolinyl}sulfanyl)-N-(4-ethylphenyl)butanamide (K284-6111), the inhibitor of CHI3L1, has the inhibitory effect on memory impairment in Αß infusion mouse model and on LPS-induced neuroinflammation in the murine BV-2 microglia and primary cultured astrocyte. METHODS: In the present study, we investigated the inhibitory effect of K284-6111 on memory dysfunction and neuroinflammation in Tg2576 transgenic mice, and a more detailed correlation of CHI3L1 and AD. To investigate the effects of K284-6111 on memory dysfunction, we administered K284-6111 (3 mg/kg, p.o.) daily for 4 weeks to Tg2576 mice, followed by behavioral tests of water maze test, probe test, and passive avoidance test. RESULTS: Administration of K284-6111 alleviated memory impairment in Tg2576 mice and had the effect of reducing the accumulation of Aß and neuroinflammatory responses in the mouse brain. K284-6111 treatment also selectively inactivated ERK and NF-κB pathways, which were activated when CHI3L1 was overexpressed, in the mouse brain and in BV-2 cells. Web-based gene network analysis and our results of gene expression level in BV-2 cells showed that CHI3L1 is closely correlated with PTX3. Our result revealed that knockdown of PTX3 has an inhibitory effect on the production of inflammatory proteins and cytokines, and on the phosphorylation of ERK and IκBα. CONCLUSION: These results suggest that K284-6111 could improve memory dysfunction by alleviating neuroinflammation through inhibiting CHI3L1 enhancing ERK-dependent PTX3 pathway.

Selection and Characterization of YKL-40-Targeting Monoclonal Antibodies from Human Synthetic Fab Phage Display Libraries.

YKL-40, also known as chitinase-3-like 1 (CHI3L1), is a glycoprotein that is expressed and secreted by various cell types, including cancers and macrophages. Due to its implications for and upregulation in a variety of diseases, including inflammatory conditions, fibrotic disorders, and tumor growth, YKL-40 has been considered as a significant therapeutic biomarker. Here, we used a phage display to develop novel monoclonal antibodies (mAbs) targeting human YKL-40 (hYKL-40). Human synthetic antibody phage display libraries were panned against a recombinant hYKL-40 protein, yielding seven unique Fabs (Antigen-binding fragment), of which two Fabs (H1 and H2) were non-aggregating and thermally stable (75.5 °C and 76.5 °C, respectively) and had high apparent affinities (KD = 2.3 nM and 4.0 nM, respectively). Reformatting the Fabs into IgGs (Immunoglobulin Gs) increased their apparent affinities (notably, for H1 and H2, KD = 0.5 nM and 0.3 nM, respectively), presumably due to the effects of avidity, with little change to their non-aggregation property. The six anti-hYKL-40 IgGs were analyzed using a trans-well migration assay in vitro, revealing that three clones (H1, H2, and H4) were notably effective in reducing cell migration from both A549 and H460 lung cancer cell lines. The three clones were further analyzed in an in vivo animal test that assessed their anti-cancer activities, demonstrating that the tumor area and the number of tumor nodules were significantly reduced in the lung tissues treated with H1 (IgG). Given its high affinity and desirable properties, we expect that the H1 anti-hYKL-40 mAb will be a suitable candidate for developing anti-cancer therapeutics.

Astrocytic IGFBP2 and CHI3L1 in cerebrospinal fluid drive cortical metastasis of HER2+breast cancer.

The brain is often reported as the first site of recurrence among breast cancer patients overexpressing human epidermal growth factor receptor 2 (HER2). Although most HER2+tumors metastasize to the subcortical region of the brain, a subset develops in the cortical region. We hypothesize that factors in cerebrospinal fluid (CSF) play a critical role in the adaptation, proliferation, and establishment of cortical metastases. We established novel cell lines using patient biopsies to model breast cancer cortical and subcortical metastases. We assessed the localization and growth of these cells in vivo and proliferation and apoptosis in vitro under various conditions. Proteomic analysis of human CSF identified astrocyte-derived factors that support the proliferation of cortical metastases, and we used neutralizing antibodies to test the effects of inhibiting these factors both in vivo and in vitro. The cortical breast cancer brain metastatic cells exhibited greater proliferation than subcortical breast cancer brain metastatic cells in CSF containing several growth factors that nourish both the CNS and tumor cells. Specifically, the astrocytic paracrine factors IGFBP2 and CHI3LI promoted the proliferation of cortical metastatic cells and the formation of metastatic lesions. Disruption of these factors suppressed astrocyte-tumor cell interactions in vitro and the growth of cortical tumors in vivo. Our findings suggest that inhibition of IGFBP2 and CHI3LI signaling, in addition to existing treatment modalities, may be an effective therapeutic strategy targeting breast cancer cortical metastasis.

Downregulated CHI3L1 alleviates skeletal muscle stem cell injury in a mouse model of sepsis.

Sepsis is an acute systemic inflammatory response of the body to microbial infection and a life-threatening condition associated with multiple organ failure. Recent data suggest that sepsis survivors present with long-term myopathy due to the dysfunction of skeletal muscle stem cells and satellite cells. Accumulating studies have implicated chitinase-3-like-1 protein (CHI3L1) in a variety of infectious diseases, specifically sepsis. Therefore, the aim of the present study is to elucidate the potential mechanism by which CHI3L1 is involved in the injury of skeletal muscle stem cells in mouse models of sepsis. An in vitro cell model was developed by lipopolysaccharide (LPS) and in vivo mouse model of sepsis was induced by CRP-like protein (CLP). To elucidate the biological significance behind the silencing of CHI3L1, modeled skeletal muscle stem cells and mice were treated with siRNA against CHI3L1 or overexpressed CHI3L1. Highly expressed CHI3L1 was found in skeletal muscle tissues of mice with sepsis. Besides, siRNA-mediated silencing of CHI3L1 was revealed to increase Bcl-2 expression along with cell proliferation, while diminishing Bax expression, cell apopstosis as well as serum levels of TNF-α, IL-1ß, INF-γ, IL-10, and IL-6. Taken conjointly, this present study provided evidence suggesting that downregulation of CHI3L1 has the potential to prevent the injury of skeletal muscle stem cells in mice with sepsis. Collectively, CHI3L1 may serve as a valuable therapeutic strategy in alleviating sepsis.

The role of tumor suppressor of resveratrol and prednisolone by downregulation of YKL-40 expression in CCRF-CEM cell line.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is characterized by excessive accumulation of lymphoblast and progenitors. Leukemia is the most common cancer in children and ALL is the most common subtype. Many studies have shown that the YKL-40 gene is one of the most widely expressed genes in tumors, including leukemia, but not in healthy blood cells. Clinical studies have shown that serum YKL-40 levels have a positive correlation with tumor expansion, in addition to being a prognostic agent independent of a short relapse-free interval, as well as a brief overall survival in patients with various cancers. The previous study shows that YKL-40 is closely related to the degree of pathology or degree of human leukemia pathology and plays an important role in cell proliferation. Hence, the YKL-40 can be an attractive target in designing anticancer therapies. METHODS: CCRF-CEM cells were treated with resveratrol and prednisolone. For analysis of YKL-40 expression changes under medication, real-time polymerase chain reaction (PCR) and Western blot techniques were used at resonating intervals of 24 and 48 hours. RESULTS: The effect of 15, 50, and 100 µM resveratrol and 700 µM of prednisolone on CCRF-CEM cells reduced YKL-40. The YKL-40 gene was quantitatively measured using RT-PCR. The Western blot method was used to evaluate changes in the expression of YKL-40 protein. CONCLUSION: In this study, we first evaluated YKL-40 expression and resveratrol and prednisolone effect on YKL-40 in ALL. This finding supports the idea of targeting YKL-40 as a new drug treatment of ALL and extends the use of resveratrol in antileukemia research.

Caffeic acid and ellagic acid ameliorate adjuvant-induced arthritis in rats via targeting inflammatory signals, chitinase-3-like protein-1 and angiogenesis.

Rheumatoid arthritis (RA) is a chronic inflammatory arthropathy that principally attacks the joints. The present study aimed to explore the potential anti-arthritic effects of caffeic acid and ellagic acid in adjuvant-induced arthritis, compared to celecoxib. The current study also explored the underlying molecular mechanisms e.g., pro-inflammatory signals including chitinase-3-like protein-1 (CHI3L1); a glycoprotein that correlates with RA joint destruction besides angiogenesis, oxidative stres and apoptosis. Interestingly, caffeic and ellagic acids attenuated the severity of arthritis with comparable efficacy to celecoxib. Both agents effectively mitigated paw edema and inflammatory cell infiltration and protected the joint tissues against pannus formation along with cartilage and bone destruction. Notably, they also lowered the paw expression of NF-κB and the downstream effector CHI3L1 and its synthesis inducer IL-1ß. They also lowered the levels of the tissue remodeling factor MMP-9 and the angiogenic signal VEGF in rat paws. Both agents also suppressed serum oxidative stress via diminishing lipid peroxides and nitric oxide together with augmentation of reduced glutathione in arthritic animals. Regarding apoptosis, they attenuated paw caspase-3 levels, favoring cell survival. Together, these favorable findings may advocate the use of caffeic and ellagic acids as adjunct modalities for the management of RA to mitigate joint damage.

Suppression of metastasis through inhibition of chitinase 3-like 1 expression by miR-125a-3p-mediated up-regulation of USF1.

Rationale: Chitinase 3-like 1 (Chi3L1) protein is up-regulated in various diseases including solid cancers. According to Genome-Wide Association Study (GWAS)/Online Mendelian Inheritance in Man (OMIM)/Differentially Expressed Gene (DEG) analyses, Chi3L1 is associated with 38 cancers, and more highly associated with cancer compared to other oncogenes such as EGFR, TNFα, etc. However, the mechanisms and pathways by which Chi3L1 is associated with cancer are not clear. In current study, we investigated the role of Chi3L1 in lung metastasis. Methods: We performed the differentially expressed gene analysis to explore the genes which are associated with Chi3L1 using the web-based platform from Biomart. We investigated the metastases in lung tissues of C57BL/6 mice injected with B16F10 melanoma following treatment with Ad-shChi3L1. We also investigated the expression of USF1 and Chi3L1 in Chi3L1 KD mice lung tissues by Western blotting and IHC. We also analyzed lung cancer cells metastases induced by Chi3L1 using migration and cell proliferation assay in human lung cancer cell lines. The involvement of miR-125a-3p in Chi3L1 regulation was determined by miRNA qPCR and luciferase reporter assay. Results: We showed that melanoma metastasis in lung tissues was significantly reduced in Chi3L1 knock-down mice, accompanied by down-regulation of MMP-9, MMP-13, VEGF, and PCNA in Chi3L1 knock-down mice lung tissue, as well as in human lung cancer cell lines. We also found that USF1 was conversely expressed against Chi3L1. USF1 was increased by knock-down of Chi3L1 in mice lung tissues, as well as in human lung cancer cell lines. In addition, knock-down of USF1 increased Chi3L1 levels in addition to augmenting metastasis cell migration and proliferation in mice model, as well as in human cancer cell lines. Moreover, in human lung tumor tissues, the expression of Chi3L1 was increased but USF1 was decreased in a stage-dependent manner. Finally, Chi3L1 expression was strongly regulated by the indirect translational suppressing activity of USF1 through induction of miR-125a-3p, a target of Chi3L1. Conclusion: Metastases in mice lung tissues and human lung cancer cell lines were decreased by KD of Chi3L1. USF1 bound to the Chi3L1 promoter, however, Chi3L1 expression was decreased by USF1, despite USF1 enhancing the transcriptional activity of Chi3L1. We found that USF1 induced miR-125a-3p levels which suppressed Chi3L1 expression. Ultimately, our results suggest that lung metastasis is suppressed by knock-down of Chi3L1 through miR-125a-3p-mediated up-regulation of USF1.

Shrimp Antiviral mja-miR-35 Targets CHI3L1 in Human M2 Macrophages and Suppresses Breast Cancer Metastasis.

Virus infection can change host's metabolism, while tumorigenesis results from metabolic disorder. MicroRNAs (miRNAs), crucial regulatory factors overlaying transcriptional control mechanisms, can guide metabolic homeostasis. In terms of metabolic disorder, antiviral miRNAs may have anti-tumor activity. However, this issue has not been extensively investigated. In the present study, the results revealed that shrimp mja-miR-35, which showed antiviral activity in shrimp against white spot syndrome virus (WSSV) infection, could suppress the metastasis of breast cancer by targeting human CHI3L1 gene of M2 macrophages in a cross-phylum manner. Furthermore, the feed expressing shrimp mja-miR-35 had antiviral capacity in shrimp and anti-tumor activity in humans, leading to the simultaneous control of virus infection and tumor progression. Therefore, our findings indicated that the antiviral miRNAs derived from shrimp stress responses against virus infection might be an important source of human anti-tumor drugs and miRNAs could bridge the control of aquaculture diseases and the prevention of human tumors.

Amelioration of atherosclerosis in apolipoprotein E-deficient mice by combined RNA interference of lipoprotein-associated phospholipase A2 and YKL-40.

To test the hypothesis that combined RNA interference (RNAi) of lipoprotein-associated phospholipase A2 (Lp-PLA2) and YKL-40 is superior to RNAi of Lp-PLA2 or YKL-40 alone in ameliorating atherosclerosis. A total of 120 apolipoprotein E-deficient mice (apoE-/- mice) were randomly divided into five groups, including the vehicle alone, scrambled RNAi, Lp-PLA2 RNAi, YKL-40 RNAi, and combined Lp-PLA2 and YKL-40 RNAi groups. Constrictive collars were used to induce plaque formation. Lp-PLA2 RNAi and YKL-40 RNAi viral suspensions were transduced into carotid plaques of the mice. Carotid plaques were harvested for histological analysis four weeks after viral vector transduction. Inflammatory gene expression in the plasma and atherosclerotic plaques was determined by ELISA and real-time PCR. Four weeks after RNAi, the serum concentration and plaque mRNA expression of Lp-PLA2 and YKL-40 were remarkably attenuated, leading to reduced inflammatory gene expression. Plaques from the Lp-PLA2 or YKL-40 RNAi group showed lower lipid content, higher collagen content, increased fibrous cap thickness, and lower mRNA expressions of MCP-1 and MMP-8 than than those in the vehicle and scramble groups. When compared with the isolated Lp-PLA2 or YKL-40 RNAi group, the combined Lp-PLA2 and YKL-40 RNAi group exhibited higher collagen content and fibrous cap thickness, and lower lipid content and local inflammation. The beneficial effects of RNAi were independent of the plasma lipoprotein profile. Combined RNAi of Lp-PLA2 and YKL-40 is superior to RNAi of Lp-PLA2 or YKL-40 alone in ameliorating atherosclerosis.

YKL-40-Induced Inhibition of miR-590-3p Promotes Interleukin-18 Expression and Angiogenesis of Endothelial Progenitor Cells.

YKL-40, also known as human cartilage glycoprotein-39 or chitinase-3-like-1, is a pro-inflammatory protein that is highly expressed in rheumatoid arthritis (RA) patients. Angiogenesis is a critical step in the pathogenesis of RA, promoting the infiltration of inflammatory cells into joints and providing oxygen and nutrients to RA pannus. In this study, we examined the effects of YKL-40 in the production of the pro-inflammatory cytokine interleukin-18 (IL-18), and the stimulation of angiogenesis and accumulation of osteoblasts. We observed that YKL-40 induces IL-18 production in osteoblasts and thereby stimulates angiogenesis of endothelial progenitor cells (EPCs). We found that this process occurs through the suppression of miR-590-3p via the focal adhesion kinase (FAK)/PI3K/Akt signaling pathway. YKL-40 inhibition reduced angiogenesis in in vivo models of angiogenesis: the chick embryo chorioallantoic membrane (CAM) and Matrigel plug models. We report that YKL-40 stimulates IL-18 expression in osteoblasts and facilitates EPC angiogenesis.

CHI3L1 regulation of inflammation and the effects on osteogenesis in a Staphylococcus aureus-induced murine model of osteomyelitis.

Osteomyelitis is an inflammation of the bone and bone marrow that occurs as a consequence of infections mainly attributed to Staphylococcus aureus. In a previous study, we found that expression of the chitinase 3-like 1 (CHI3L1) gene affected mineralization of MC3T3-E1 cells infected with S. aureus and there was increased expression of CHI3L1 in the blood of osteomyelitis patients. In the present study, to further investigate the role of CHI3L1 in osteomyelitis, we developed an S. aureus-induced murine model of the disease. We found that the expression of CHI3L1 was significantly up-regulated in femurs of mice infected with S. aureus compared with mice inoculated with a PBS control. To investigate these results further, we performed a CHI3L1 knock-down by lentivirus-mediated RNA interference in mice. Micro-computed tomography of infected femurs revealed that S. aureus triggers profound alterations in bone turnover, and femurs of CHI3L1 short hairpin RNA (shRNA-CHI3L1)-injected mice infected with S. aureus have significantly less cortical bone destruction when compared with control mice infected with S. aureus. Inhibition of CHI3L1 also decreased inflammation by reducing levels of proinflammatory cytokines and promoted the process of osteogenesis. The Notch signaling pathway has been shown to play an important role in modulating the differentiation of osteoblasts and osteoclasts. Our study showed that Notch1, Jagged1 and Hes1 expression significantly decreased in mice infected with S. aureus compared with the control, and shRNA-CHI3L1 could increase their level in S. aureus-infected mice. This research indicates that inhibition of CHI3L1 can reduce the debilitating effects of S. aureus in a murine model of osteomyelitis.

MicroRNA-24 Modulates Staphylococcus aureus-Induced Macrophage Polarization by Suppressing CHI3L1.

Macrophages play a crucial role in host innate anti-Staphylococcus aureus defense, which is tightly regulated by multiple factors, including microRNAs. A recent study showed that miR-24 plays an important role in macrophage polarization. Here, we investigated the biological function of miR-24 in S. aureus-stimulated macrophages. The results revealed that miR-24 expression was significantly decreased in both human and mouse macrophage cell lines with S. aureus stimulation in a time-dependent manner. Moreover, miR-24 overexpression significantly decreased the production of M1 phenotype markers, such as IL-6, iNOS, TNF-α, CD86, and CD80, whereas it increased the production of M2 markers, such as Arg1, CCL17, CCL22, CD163, and CD206, in S. aureus-stimulated macrophages. Conversely, knockdown of miR-24 promoted M1 macrophage polarization but diminished M2 macrophage polarization in S. aureus-stimulated macrophages. Furthermore, CHI3L1 was predicted as a target gene of miR-24 using bioinformatics software and identified by luciferase reporter assay. Additionally, miR-24 overexpression inhibited CHI3L1 expression and downregulated the downstream MAPK pathway in S. aureus-stimulated macrophages. Finally, CHI3L1 overexpression rescued macrophage polarization and MAPK pathway inhibition induced by miR-24 mimic transfection in S. aureus-stimulated macrophages. In conclusion, the data suggest that miR-24 serves as a molecular regulator in S. aureus-induced macrophage polarization through targeting of CHI3L1 and regulation of the MAPK pathway, which may provide a promising therapeutic target for S. aureus-related infections and inflammatory diseases.

Inhibition of Mammalian Glycoprotein YKL-40: IDENTIFICATION OF THE PHYSIOLOGICAL LIGAND.

YKL-40 is a mammalian glycoprotein associated with progression, severity, and prognosis of chronic inflammatory diseases and a multitude of cancers. Despite this well documented association, identification of the lectin's physiological ligand and, accordingly, biological function has proven experimentally difficult. YKL-40 has been shown to bind chito-oligosaccharides; however, the production of chitin by the human body has not yet been documented. Possible alternative ligands include proteoglycans, polysaccharides, and fibers like collagen, all of which makeup the extracellular matrix. It is likely that YKL-40 is interacting with these alternative polysaccharides or proteins within the body, extending its function to cell biological roles such as mediating cellular receptors and cell adhesion and migration. Here, we consider the feasibility of polysaccharides, including cello-oligosaccharides, hyaluronan, heparan sulfate, heparin, and chondroitin sulfate, and collagen-like peptides as physiological ligands for YKL-40. We use molecular dynamics simulations to resolve the molecular level recognition mechanisms and calculate the free energy of binding the hypothesized ligands to YKL-40, addressing thermodynamic preference relative to chito-oligosaccharides. Our results suggest that chitohexaose and hyaluronan preferentially bind to YKL-40 over collagen, and hyaluronan is likely the preferred physiological ligand, because the negatively charged hyaluronan shows enhanced affinity for YKL-40 over neutral chitohexaose. Collagen binds in two locations at the YKL-40 surface, potentially related to a role in fibrillar formation. Finally, heparin non-specifically binds at the YKL-40 surface, as predicted from structural studies. Overall, YKL-40 likely binds many natural ligands in vivo, but its concurrence with physical maladies may be related to associated increases in hyaluronan.