Neutrophils trap and kill bacteria by forming highly decondensed chromatin structures, termed neutrophil extracellular traps (NETs). We previously reported that histone hyper citrullination catalyzed by peptidylarginine deiminase 4 (PAD4) correlates with chromatin decondensation during NET formation. However, the role of PAD4 in NETmediated bacterial trapping and killing has not been tested. Here, we use PAD4 knockout mice to show that PAD4 is essential for NETmediated antibacterial function. Unlike PAD4+/+ neutrophils, PAD4/ neutrophils cannot form NETs after stimulation with chemokines or incubation with bacteria, and are deficient in bacterial killing by NETs. In a mouse infectious disease model of necrotizing fasciitis, PAD4/ mice are more susceptible to bacterial infection than PAD4+/+ mice due to a lack of NET formation. Moreover, we found that citrullination decreased the bacterial killing activity of histones and nucleosomes, which suggests that PAD4 mainly plays a role in chromatin decondensation to form NETs instead of increasing histonemediated bacterial killing. Our results define a role for histone hypercitrullination in innate immunity during bacterial infection.

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