ExoU, a virulence factor with recognizable importance in invasive acute infections, stimulates a proinflammatory response that is characterized, among other events, by a robust neutrophil infiltrate 
. The early recruitment of neutrophils is a crucial mechanism of defense against P. aeruginosa
, as proved by the increased mortality of mice submitted to neutrophil depletion before infection and the higher disease severity of neutropenic patients 
. However, excessive and continuous neutrophil infiltration and activation is a harmful event because highly reactive intermediates and degradative enzymes secreted by these cells accumulate in tissues, promoting organ damage that could facilitate bacterial spread. Furthermore, ExoU kills neutrophils 
, releasing intracellular products that induce even more inflammation and injury. Hence, by recruiting and killing neutrophils, ExoU could interfere with immune response, promoting a local tissue damage that would favor bacterial dissemination.
The mechanisms regulated by ExoU that lead to neutrophil infiltration are still poorly understood and, although P. aeruginosa
stimulates ICAM-1 expression in endothelial cell surface, there is no evidence of ExoU contribution to ICAM-1 up-regulation 
. Here, we show that the ExoU-mediated neutrophil recruitment results, at least in part, from the higher IL-8 expression due to the ExoU-dependent NF-κB activation. This finding is of special interest because NF-κB is a transcriptional factor that regulates a number of genes involved in inflammatory and immune responses and can represent a potential target in P. aeruginosa
Although IL-8 stimulation by ExoU in cell culture assays has been previously reported 
, induction of IL-8 expression had only been related to AP-1 pathway. In fact, IL-8 expression could simultaneously be regulated by multiple transcriptional factors and, in a model of chronic infection during cystic fibrosis, a non-producing ExoU P. aeruginosa
strain stimulated IL-8 secretion through a complex mechanism that included activation of NF-κB, AP-1, NF-IL6, CHOP and CREB 
. However, in our model, the treatment of cells with a combination of JNK and NF-κB inhibitors did not reduce IL-8 secretion even more than the observed in cells treated with NF-κB inhibitor only. Furthermore, the ExoU-mediated activation of NF-κB pathway is probably a common event since our data show that it occurs in both epithelial and endothelial cells. More importantly, we show that the ExoU-mediated KC secretion and neutrophil recruitment during acute pneumonia was profoundly affected by NF-κB inhibition, which demonstrate the central role of this transcriptional factor in the proinflammatory response induced by ExoU.
Additionally, IL-8 transcription is affected by modulation of epigenetic patterns that regulates chromatin opening and, consequently, the access of transcription factors to the promoter region. A number of studies showed that bacteria can regulate IL-8 gene by inducing or blocking histone modifications, such as histone phosphorylation, methylation or acetylation 
Since the transcriptional activation domain of p65 interacts with histone acetyltransferases 
, ExoU activation of NF-κB could induce epigenetic alterations that would contribute to the increased expression of IL-8. The ability of ExoU or other P. aeruginosa
PAMPs to induce histone modifications during infection of host cells by ExoU-producing strains could represent a promising field of research that remains to be explored.
Bacterial products can bind to cellular receptors, such as P. aeruginosa
flagellin that binds to TLR5, and directly activate NF-κB, leading to increased IL-8 expression 
. On the other hand, virulence factors generate a number of cellular responses and lead to the secretion of host products that in turns can activate NF-κB and induce IL-8 secretion. This is an interesting question that remains to be solved. ExoU directly activates NF-κB or this effect is resulted from products generated by ExoU-injected cells that could act in all cells of the neighborhood?
Similar to other PLA2
enzymes, ExoU acts in host membranes phospholipids, releasing free arachidonic acid and lysophospholipids, which induce a number of pathways that lead to NF-κB activation. We have previously reported that ExoU, probably by arachidonic acid oxidation or stimulus of NADPH oxidase, causes oxidative stress in P. aeruginosa
-infected cells 
. Oxidative stress is widely considered a potent NF-κB activator and it was shown to drive NF-κB-dependent IL-8 expression in a variety of studies 
. Additionally, free arachidonic acid is also metabolized by COX-2, leading to higher PGE2
secretion in ExoU-injected cells 
. Since PGE2
can activate NF-κB and induce IL-8 expression in epithelial endometrial cells, this mechanism could represent an interesting via to be explored as well 
. Besides arachidonic acid, infection with ExoU-expressing P. aeruginosa
strains also lead to release of lysophospholipids, which can be acetylated to produce platelet activating factor (PAF), a potent inflammatory lipid. In fact, we have previously demonstrated that ExoU induces the production of PAF 
, and this compound is known to be able to activate NF-κB and stimulate IL-8 secretion 
. Finally, diverse other products generated in response to ExoU could activate NF-κB or sustain this activation, such as TNF-α 
that classically activates NF-κB upon binding to TNFR family members 
In conclusion, our study showed that NF-κB is an important target explored by ExoU to modulate host response. Regulation of NF-κB pathway could be used to control the inflammatory response and avoid the injury provoked by the excessive neutrophil infiltration and the products secreted by these cells or released during the ExoU-induced cell death. Moreover, by activating NF-κB, ExoU probably regulates a wide range of genes that participate of diverse cellular processes, which turns this pathway an attractive focus to be investigated. Currently, further studies are being performed in our laboratory to identify the signaling pathways that lead to NF-κB activation by ExoU.