Objectives: Staphylococcus aureus is a major cause of skin, soft tissue and severe systemic infections. The emergence of methicillin-resistant Staphylococcus aureus (MRSA) has further increased the risk of mortality. Therefore, development of adjunct therapies targeting the host immune response becomes important. The innate immune response against S. aureus is substantially mediated by macrophages and involves the NF-κB pathway. The deubiquitinating enzyme CYLD negatively regulates the NF-κB pathway by cleaving K-63 linked polyubiquitin chains from several signaling molecules. This project aims to investigate how CYLD regulates the outcome of S. aureus infection in murine and human macrophages.

Methods: Wild-type (WT) and Cyld -/- mice were intravenously infected with S. aureus and the weight loss was monitored daily until day 49 p.i.. The bacterial loads in liver, kidneys and spleen were quantified. To delineate the role of CYLD in macrophages, bone marrow-derived macrophages (BMDM) from WT and Cyld-/- mice were stimulated in vitro with IFN-g and LPS for 24 hours followed by infection with S. aureus. The bacterial load in the macrophages was enumerated 24 hours p.i. To identify the underlying CYLD-regulated signaling pathways, the activation of NF-κB, ERK1/2, and p38MAPK was analyzed by WB in S. aureus infected BMDM and MRSA-infected THP-1 macrophages. Furthermore, CYLD was deleted in human THP-1 cells by CRISPR-Cas9 and differentiated into M0, M1 and M2 macrophages. The bacterial load in WT and Cyld-/- THP-1 macrophages was enumerated after infection with MRSA.

Results: CYLD-deficiency reduced bacterial burden in S. aureus infection both in vivo and in vitro. Additional in vitro experiments using murine BMDM and human THP-1 derived macrophages showed enhanced activation of NF-κB, p38MAPK and ERK1/2 pathways, and increased production of anti-bacterial antibacterial ROS in CYLD deficient macrophages leading to better control of S. aureus.

Conclusions: Our study identifies CYLD as an inhibitor of anti-staphylococcus immune responses and a potential therapeutic target to treat S. aureus infections.