Of relevance in this context, there is still the possibility of other transcription factors which have been reported in udder infections such as activator protein-1, or peroxisome proliferator-activated receptors, or regulatory factors such as CAAT box enhancer binding proteins. Regardless the underlying transcription mechanism, our data overall imply that proIL-1beta is both highly and differentially processed within 12 h to 24 h post-IMI with both mastitis pathogens, which suggests its tight regulation. The key function of IL-1beta is highlighted by its ability to control the bacterial growth of Gram-negative and to a lesser extend also Grampositive bacteria. Moreover, the revealed maturation process is certainly also non-classical as it occurs independently from the classical caspase-1 pathway. As a differential IL-1beta fragmentation pattern was observed after Gram-negative compared to Gram-positive mastitis, the cytokine protein profile data from the current study support the hypothesis that E. coli and S. aureus overall induce a markedly different IL-1beta activity. Our data also corroborate previous findings in KO mice demonstrating that neutrophil IL-1R signaling in mammary gland inflammation mediates neutrophil BIBW2992 EGFR/HER2 inhibitor influx from the interstitium and capillaries into the lumen of the alveoli. Additionally these innate immune cells restrict E. coli invasion into the mammary epithelial cells, which is only a characteristic of S. aureus infections in wild type mice. We hypothesize that proIL-1beta is locally secreted by the mammary epithelial cells and that multiple fragments likely result from the molding of bacterial proteases and/or NF-kappaB-induced non-caspase proteases secreted from neutrophils and mammary epithelial cells. Following infection with both pathogens, the IL-1beta proform is likely present in the interstitium where it is cleaved by serine proteases produced by NF-kappaB activity in neutrophils, which are already sensitized by the preceding release of other proinflammatory cytokines following epithelial NF-kappaB signaling. Upon E. coli infection in mice, the host rapidly induces an efficient protective program that involves mammary NF-kappaB transcription to enhance TNF-alpha. Interestingly, TNF-alpha is a well-known inducer of MMPs, which subsequently accelerates mammary gland involution. Subsequently, proIL-1beta becomes inactivated by additional cleavage into fragments abolishing an additional neutrophil influx into the lumen but increasing the number of shedded epithelial cells in order to restrict bacterial growth as well as neutrophil cytotoxicity. This explains the lower IL-1beta levels at 24 h post-IMI versus 12 h post-IMI with E. coli. In marked contrast, NF-kappaB is also rapidly but far less activated after S. aureus and thus a delayed influx of neutrophils occurs. Moreover, significantly less TNF-alpha is released and thus MMPs will not cause their typical IL-1beta cleavage patterns. In contrast to the early neutrophil-mediated E. coli mediated immune response, our data are indicative for the activity of S. aureus-derived proteases that alternatively cleave proIL-1beta enabling the typical evasion of these Gram-positive pathogens from the alveolar lumen.