Screening for C-type lectin receptor (CLR)/bacteria interactions using a bovine CLR-Fc fusion protein library reveals recognition of Pasteurella multocida B:2 by MICL

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.