Chitin-mediated blockade of chitinase-like proteins reduces tumor immunosuppression, inhibits lymphatic metastasis and enhances anti-PD-1 efficacy in complementary TNBC models.

BACKGROUND: Chitinase-like proteins (CLPs) play a key role in immunosuppression under inflammatory conditions such as cancer. CLPs are enzymatically inactive and become neutralized upon binding of their natural ligand chitin, potentially reducing CLP-driven immunosuppression. We investigated the efficacy of chitin treatment in the context of triple-negative breast cancer (TNBC) using complementary mouse models. We also evaluated the immunomodulatory influence of chitin on immune checkpoint blockade (ICB) and compared its efficacy as general CLP blocker with blockade of a single CLP, i.e. chitinase 3-like 1 (CHI3L1). METHODS: Female BALB/c mice were intraductally injected with luciferase-expressing 4T1 or 66cl4 cells and systemically treated with chitin in combination with or without anti-programmed death (PD)-1 ICB. For single CLP blockade, tumor-bearing mice were treated with anti-CHI3L1 antibodies. Metastatic progression was monitored through bioluminescence imaging. Immune cell changes in primary tumors and lymphoid organs (i.e. axillary lymph nodes and spleen) were investigated through flow cytometry, immunohistochemistry, cytokine profiling and RNA-sequencing. CHI3L1-stimulated RAW264.7 macrophages were subjected to 2D lymphatic endothelial cell adhesion and 3D lymphatic integration in vitro assays for studying macrophage-mediated lymphatic remodeling. RESULTS: Chitin significantly reduced primary tumor progression in the 4T1-based model by decreasing the high production of CLPs that originate from tumor-associated neutrophils (TANs) and Stat3 signaling, prominently affecting the CHI3L1 and CHI3L3 primary tumor levels. It reduced immunosuppressive cell types and increased anti-tumorigenic T-cells in primary tumors as well as axillary lymph nodes. Chitin also significantly reduced CHI3L3 primary tumor levels and immunosuppression in the 66cl4-based model. Compared to anti-CHI3L1, chitin enhanced primary tumor growth reduction and anti-tumorigenicity. Both treatments equally inhibited lymphatic adhesion and integration of macrophages, thereby hampering lymphatic tumor cell spreading. Upon ICB combination therapy, chitin alleviated anti-PD-1 resistance in both TNBC models, providing a significant add-on reduction in primary tumor and lung metastatic growth compared to chitin monotherapy. These add-on effects occurred through additional increase in CD8α+ T-cell infiltration and activation in primary tumor and lymphoid organs. CONCLUSIONS: Chitin, as a general CLP blocker, reduces CLP production, enhances anti-tumor immunity as well as ICB responses, supporting its potential clinical relevance in immunosuppressed TNBC patients.

Chitinase1 contributed to a potential protection via microglia polarization and Aß oligomer reduction in D-galactose and aluminum-induced rat model with cognitive impairments.

Chitinase activity is increased in Alzheimer's disease (AD). However, the role of chitinase1 in AD is unknown. We investigated the effects of chitinase1 on Alzheimer's pathology and microglia function. Artificial chitinase1 and chitinase inhibitor (chitinase-IN-2) were used to determine the effects of chitinase1 on inflammatory factors and ß-amyloid (Aß) oligomers deposition in D-galactose/AlCl3-induced rat model with cognitive impairments. Aß-treated N9 microglia cells were analyzed to further verify whether the changes in inflammatory factors following chitinase1 treatment were associated with microglia alternative activation. Our data displayed that the activity of chitinase1 was both improved in D-galactose/AlCl3-injected rats and Aß-pretreated microglia. Moreover, there was an improvement in cognitive function in chitinase1-treated AD rats. Furthermore, anti-inflammation factors (Arginase 1, Arg-1, mannose receptor type C 1, MRC1/CD206) were increased and pro-inflammation factors (tumor necrosis factor alpha, TNFα, interleukin 1 beta, IL-1ß) were decreased in D-galactose/AlCl3-induced AD rats with chitinase1 treatment. A higher level of M2 markers (Arg-1, MRC1/CD206) and a lower level of classic M1 markers (TNFa, IL-1ß) were obtained in Aß-pretreated N9 cells with chitinase1, suggesting that chitinase1 polarized the microglia into an anti-AD M2 phenotype. We also detected that chitnase1 could weaken the deposition of Aß oligomers in the brain of D-galactose/ AlCl3-induced AD rats. In conclusion, Chitinase1 might exert protective effects against AD by polarizing microglia to an M2 phenotype and resisting Aß oligomer deposition.

The influence of carbohydrases on the growth of fungal pathogens in vitro and in vivo.

Mixtures of mycolytic enzymes from various sources release protoplasts from living fungal tissue under suitable conditions. Such enzyme mixtures obtained from Coprinus comatus (mycolase I), Physarum polycephalum (mycolase II) and Lycoperdon pyriforme (mycolase III) are of low toxicity in mammals when given parenterally and are able to cure experimental systemic fungal infections in mice when administered alone or in conjunction with normally ineffective levels of conventional antimycotic drugs such as amphotericin B. The effect is believed to be due to enzymic degradation of the fungal cell wall either killing the fungus directly or enhancing activity of existing antifungal agents by increasing access to the cell interior.