CCAAT/Enhancer Binding Protein Epsilon Mediates Nicotinamide-Enhanced Clearance of Staphylococcus Aureus Infection

Abstract 925

Staphylococcus aureus in community and healthcare settings commonly causes serious and potentially life-threatening infections. Widespread use of antibiotics is responsible for the emergence and rapid spread of resistant pathogens, including methicillin-resistant S. aureus (MRSA), and highlights a pressing need for development of novel antimicrobial therapies. The myeloid-specific transcription factor, CCAAT/enhancer binding protein epsilon (C/EBPε) serves as a critical regulator of the terminal differentiation and functional maturation of neutrophils and macrophages, both crucial components of the innate immune system.

Comparable to humans with neutrophil specific granule deficiency (SGD) carrying a causative mutation in this transcription factor, we showed that C/EBPε-deficient (C/EBPε^—/—) mice were severely affected by in vivo infection with S. aureus. Paradoxically, depletion of the defective neutrophils attenuated disease pathology and even improved the outcome of infection. During subcutaneous infection with S. aureus, C/EBPε^—/— mice treated with mouse anti-polymorphonuclear neutrophil antibody showed significantly smaller skin lesions, fewer CFU within the lesion, and reduced systemic spread of bacteria. In addition, whole blood from C/EBPε^—/— mice was less effective at killing S. aureus compared to their cell-free plasma. Therefore, ineffective clearance of S. aureus by C/EBPε^—/— neutrophils, even compared to extracellular killing mechanisms, likely permitted S. aureus to thrive within neutrophils, which further aggravated the infection.

Because C/EBPε plays a critical role in the host immune response against S. aureus infection, we further hypothesized that increased activity of C/EBPε could enhance immune killing of bacteria. Using a zink-inducible expression vector, we induced overexpression of C/EBPε in U937-macrophages, and thereby enhanced bacterial clearance including MRSA by up to 1.5 log₁₀ CFU/mL. Interestingly, we found that the epigenetic modulator, nicotinamide (NAM; vitamin B3), increased activity of C/EBPε as well as downstream antimicrobial targets. Upon exposing bone-marrow derived macrophages or mononuclear cells from wildtype mice to NAM (1 or 10 mM), increased levels of lysine acetylation on core histone H3 were detected at the promoter region of CEBPE. This was associated with elevated mRNA and protein levels of C/EBPε, and increased expression of downstream antimicrobials such as cathelicidin(-related) antimicrobial peptide (CAMP) and Lactoferrin. In an in vitro, as well as in vivo infection model, moderately concentrated NAM enhanced killing of S. aureus by up to 3 log₁₀, but had no effect when administered to C/EBPε-deficient mice. This again points to C/EBPε as an important target to boost killing of bacteria by the innate immune system. Strikingly, and consistent with our murine data, NAM treatment reduced the ability of S. aureus to survive in whole human blood obtained from 12 healthy humans by 2–3 log₁₀. In line with our findings on S. aureus, we were able to demonstrate similar immune boosting effects of NAM in human blood infected with other important human pathogens such as K. pneumoniae and P. aeruginosa.

In an age when the number of antibiotics in the pipeline is limited and development of resistance occurs rapidly, use of complementary strategies to antibiotic treatment provides a promising method of limiting development of antibiotic resistance. Here, we demonstrated that C/EBPε is a regulatory factor that critically impacts the host's ability to fight bacterial infections. Compounds exerting modulatory effects on this myeloid-specific transcription factor may emerge as important antimicrobial therapeutics against frequent pathogens such as S. aureus.