Host chitinase CHIT1 generates oligomeric chitin MAMPs from pathogenic fungi for CD14-TLR1-TLR2 activation

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Dokumentart: PhDThesis
Date: 2022-04-20
Language: English
Faculty: 4 Medizinische Fakultät
Department: Medizin
Advisor: Weber, Alexander (Prof. Dr.)
Day of Oral Examination: 2022-02-15
DDC Classifikation: 610 - Medicine and health
Other Keywords:
Candida albicans
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Chitin (N-acetyl-glucosamine) is a highly abundant polysaccharide in nature and a major component of fungal cell walls, insects and common house dust allergens. Chitin also serves as microbe-associated molecular patterns (MAMPs) which is recognized by pattern recognition receptors (PRRs) of the innate immune system. In our previous study, we demonstrated that chitin oligomers longer than 6 units induce an inflammatory response via the PRR Toll-like receptor (TLR) 2. However, it remained unclear whether the co-receptors and accessory proteins are required for sensing and how chitin oligomers could be released from organisms containing polymeric chitin, which is considered immunologically inert. Interestingly, mammals express chitotriosidase-1 (CHIT1), namely chitinase, which has been determined as a biomarker constitutively expressed in fungal infection, cystic fibrosis and allergic asthma. However, the detailed role of CHIT1 in mediating innate immunity that correlates with its chitinolytic function has not been well understood. Thus, with this knowledge, it was hypothesized that host-chitinases like CHIT1 mediate the degradation of chitin-rich organisms to generate immunogenic oligomeric chitin, whose recognition is mediated by TLR2-expressing innate immune cells. Firstly, by examining two human chitotriosidase isoforms of 50 kDa and 39 kDa, we found that CHIT1 39 kDa isoform degradation products of chitin flakes, Candida albicans and house dust mites could induce TLR2-dependent NF-kB activation. Importantly, this immunogenic effect of CHIT1 39 kDa isoform required its intact catalytic activity. Furthermore, we found that chitin sensing of chitin particles and chitin-rich organisms might not be strictly dependent on direct contact, but could be mediated by diffusible oligomers generated in the presence of CHIT1. In addition, by using size exclusion chromatography (SEC) and mass spectrometric (MS) to prepare defined units of chitin oligomers, we found that optimally 14 - 16 units long oligomers triggered TLR2-NF- kB dependent inflammatory responses. To elucidate the involvement of TLR2 co- receptors, analysis in bimolecular complementary fluorescence (BiFC) assays and Tlr- deficient murine macrophages were performed and showed that chitin oligomers induce TLR1 and TLR2 heterodimerization to trigger NF-kB signaling and subsequently the production of pro-inflammatory cytokines. Finally, we demonstrated that CD14 and lipopolysaccharide binding protein (LBP), known mediators in the sensing of other hydrophobic MAMPs, enhanced and facilitated TLR2-NF-kB dependent chitin sensing. In overall, our study not only highlights CHIT1 as a novel therapeutic target for chitin- related inflammation disease but reveals an intricate system of MAMP generation, PRR sensing and induction of an enzymatic MAMP “generase” that bears resemblance to MAMP sensing in invertebrates.

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