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Published on: October 15, 2016
Screening for C-type lectin receptor (CLR)/bacteria interactions using a bovine CLR-Fc fusion protein library reveals
Samira Christin Görig1, Yeliz Gün2, Dimitri Leonid Lindenwald1
1Institute for Immunology, University of Veterinary Medicine Hannover, Bünteweg 2, 30559, Hannover, Germany; Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine, Bünteweg 17, 30559, Hannover, Germany.
Abstract:
Pattern recognition receptors (PRRs) are an essential component of the innate immune system. Myeloid C-type-lectin receptors (CLRs) serve as PRRs and play a crucial role in pathogen recognition. While the role of CLRs has been mainly studied in mice and humans, their function in cattle is poorly understood. To address this gap, we generated a novel bovine CLR-hFc fusion protein library, enabling high-throughput screening of bovine CLR/pathogen interactions. The functionality of the bovine CLR-hFc fusion proteins was validated with known CLR ligands using ELISA- and flow cytometry-based binding assays, by comparison of bovine CLRs with their murine, ovine and human orthologues. In a proof-of-principle pathogen binding study, we assessed CLR binding to Pasteurella (P.) multocida, a Gram-negative bacterial pathogen causing hemorrhagic septicemia in cattle. The bovine CLR myeloid inhibitory C-type lectin (MICL, Clec12A) was identified as a potential receptor for P. multocida, as it exhibited significant binding in flow cytometry binding assays. Cross-species analysis confirmed that murine and ovine MICL also binds P. multocida, suggesting an evolutionarily conserved recognition. To explore MICL-dependent innate responses to P. multocida-derived factors, cytokine assays were performed using dendritic cells (DCs) from wild-type (WT) and MICL-deficient (MICL-/-) mice. MICL-/- DCs produced higher levels of IL-6 and IL-12 upon stimulation with heat-killed P. multocida, suggesting a role for MICL in the down-modulation of innate responses. The results highlight MICL as a receptor in the recognition of P. multocida and demonstrate the utility of the generated bovine CLR-hFc fusion protein library for pathogen screening.
Insights
Researchers identified bovine myeloid inhibitory C-type lectin (MICL) as a receptor for Pasteurella multocida. This finding, using a novel bovine C-type lectin receptor (CLR) library, suggests MICL down-regulates innate immune responses to this cattle pathogen.
Area of Science:
- Immunology
- Infectious Disease
Background:
- Pattern recognition receptors (PRRs), including C-type lectin receptors (CLRs), are vital for innate immunity and pathogen recognition.
- While CLR functions are well-studied in mice and humans, their role in cattle immunity remains largely unexplored.
Purpose of the Study:
- To investigate the function of bovine CLRs in pathogen recognition.
- To develop and utilize a novel bovine CLR-hFc fusion protein library for high-throughput screening of CLR-pathogen interactions.
- To identify specific CLRs involved in the recognition of *Pasteurella multocida*, a significant cattle pathogen.
Main Methods:
- Generation and validation of a bovine CLR-hFc fusion protein library using ELISA and flow cytometry.
- Assessment of CLR binding to *Pasteurella multocida* using flow cytometry.
- Cross-species comparative analysis of CLR binding.
- Cytokine assays using dendritic cells from wild-type and MICL-deficient mice to evaluate MICL-dependent responses.
Main Results:
- The bovine myeloid inhibitory C-type lectin (MICL, Clec12A) was identified as a significant binding receptor for *Pasteurella multocida*.
- Cross-species analysis revealed that murine and ovine MICL also bind *P. multocida*, indicating conserved recognition.
- MICL-deficient dendritic cells showed elevated IL-6 and IL-12 production upon stimulation with heat-killed *P. multocida*, suggesting MICL's role in down-regulating innate immune responses.
Conclusions:
- Bovine MICL acts as a receptor for *Pasteurella multocida*, contributing to cattle innate immunity.
- The developed bovine CLR-hFc fusion protein library is a valuable tool for screening CLR-pathogen interactions.
- MICL appears to play a role in modulating the innate immune response to *P. multocida*.
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