Neutrophils are the main proinflammatory cell type in chronically infected lungs of cystic fibrosis (CF) patients; however, they fail to effectively clear the colonizing pathogens. Here, we investigated the molecular composition of non-mucoid and mucoid Pseudomonas aeruginosa-induced neutrophil extracellular traps (NETs) in vitro and compared them to the DNA-protein complexes present in the CF sputum. The protein composition of P. aeruginosa-induced NET fragments revealed that irrespective of the inducing stimuli, NET fragments were decorated with a conserved set of proteins. The DNA-protein complexes derived from CF sputum were consistent with NETosis and shared a similar protein signature, suggesting that the majority of the extracellular DNA was NET derived. The ability of polymorphonuclear leukocytes to produce NETs in response to P. aeruginosa was driven by macrophage migration-inhibitory factor (MIF) by promoting mitogen-activated protein kinase. Analysis of 132 CF patient samples revealed that elevated MIF protein levels correlated with poorer lung function. We suggest that targeting MIF by small molecular inhibitors might reduce the presence of extracellular DNA and serve as an adjunct to the use of antimicrobial drugs that could ultimately reduce bacterial fitness in the lungs during the later stages of CF disease.

Ocular bacterial keratitis, often associated with Pseudomonas aeruginosa bacterial infection, commonly occurs in contact lens wearers and may lead to vision impairment. In this study, we analyzed the contribution of neutrophil extracellular traps (NETs) to the mediation of protection during ocular keratitis. Both invasive and cytotoxic P. aeruginosa clinical isolates induced NET release by neutrophils. NETs carried the characteristic histone proteins, elastase, lysozyme, myeloperoxidase, and metabolic enzymes. While the invasive P. aeruginosa strains PAO1 (serogroup O5) and 6294 (serogroup O6) were trapped by NETs, the cytotoxic P. aeruginosa strains 6077, 6206 (serogroup O11), and PA14 (serogroup 010) were less sensitive to NET capture. The mechanism of escape by the cytotoxic strains from adhesion to NETs involved the shedding of outer membrane vesicles (OMVs) that outcompeted the cytotoxic P. aeruginosa strains for NET binding. When ocular infection was caused by an invasive strain in vivo, NETs were released at the ocular surface to capture bacteria, limiting their spread. Treatment with MNase I had a dose-dependent effect, with low doses of MNase speeding up bacterial clearance and high doses of MNase having toxic consequences. Cumulatively, our data suggest that NET-mediated immunity is a two-step process. Initially, pathogens attach to NET fragments; subsequently, upon nuclease activity, active serine proteases, which proteolytically degrade NET-associated proteins and promote DNase activity, are released. Therefore, a balance between NET production and NET degradation is needed to achieve maximal NET immunity. Infections caused by Pseudomonas aeruginosa are frequently associated with bacterial ocular keratitis, a condition that carries the risk of vision impairment and occurs in contact lens wearers or after ocular trauma. The majority of bacterial clinical isolates derived from corneal ulcers arising during keratitis can be divided into two functionally distinct groups: invasive and cytotoxic isolates. These two groups have distinct phenotypes; the cytotoxic strains cause rapid cell lysis, whereas the invasive strains replicate in the target cells (1, 2). These differences translate into various severities of disease; in mouse keratitis studies where infection is induced by cytotoxic strains, disease pathology is worse than that caused by invasive strains (3). Consistent with these findings, patients infected with cytotoxic strains have experienced more severe pathology and, consequently, suffered from greater visual impairment after recovery from infection (4). The immune response to both invasive and cytotoxic strains is dominated by dense neutrophil infiltrates, yet the infiltration pattern and responses of polymorphonuclear neutrophils (PMNs) differ depending on the type of strain. Due to the recently described ability of neutrophils to release DNA in the form of neutrophil extracellular traps (NETs) (5) and the presence of extracellular DNA during cytotoxic infections, we questioned whether the cyt...

Expression of SH 2 -homology-containing protein-tyrosine phosphatase-1 (SHP-1), a candidate tumor suppressor, is repressed in human T-cell leukemia virus type-1 (HTLV-1)-transformed lymphocyte cell lines, adult T-cell leukemia (ATL) cells, and in other hematologic malignancies. However, the mechanisms underlying regulation and repression of SHP-1 remain unclear. Herein, we cloned the putative full-length, hematopoietic cellspecific SHP-1 P2 promoter and identified the "core" promoter regions. HTLV-1 Tax profoundly represses P2 promoter activity and histone deacetylase-1 (HDAC1) potentiates such inhibition. NF-B was implicated as both a rate-limiting factor for basal P2 promoter activity and important for Tax IntroductionAdult T-cell leukemia (ATL) is an aggressive malignancy of CD4 ϩ , CD25 ϩ T cells, and the human T-cell leukemia virus type-1 (HTLV-1) has been identified as the causative agent. 1,2 While the understanding of ATL pathogenesis currently remains incomplete, the HTLV-1 virusencoded Tax protein has been implicated as a major contributor in the development of ATL. [3][4][5][6] The oncogenic potential of HTLV-1 Tax has been associated with its ability to modulate expression and function of cellular targets involved in cell proliferation and differentiation. 7,8 For example, Tax has been shown to induce the activation of NF-B, CREB, AP-1, and SRF 6 as well as to up-regulate IL-2/IL-2 receptor-␣, 9 IL-15, 10 IL-4, 11 12 and OX40/OX40L 6 pathways, resulting in the stimulation of cell growth. Tax can also be a negative regulator of gene expression/function and can down-regulate expression of genes involved in host DNA repair, 13 maintaining genetic stability 14 and cell cycle progression. 15 Of particular importance to this study, Tax has been shown to exert negative effects on at least 3 cellular tumor suppressorsRb, [16][17][18][19] hDLG, a human homolog of the Drosophila discs large tumor suppressor protein, 6,20-22 and p53. 6,23,24 Tax alone is able to immortalize primary human T lymphocytes and transform rodent fibroblast in vitro. 25,26 Transgenic mice expressing Tax can also develop tumor in vivo with a wide range of phenotypes . 25,27,28 Among the cellular dysfunctions caused by HTLV-1 infection, the loss of IL-2 dependence is remarkable in many HTLV-1-transformed cells. 29 In HTLV-1-infected cord blood lymphocytes, the transition from IL-2-dependent to IL-2-independent growth has been shown to correlate with the acquisition of a constitutively activated Jak/STAT pathway, suggesting the involvement of this pathway in HTLV-1-mediated T-cell transformation. 30 In addition, proliferation of uncultured leukemic cells from ATL patients has been reported to be associated with constitutive activation of Jak/STAT proteins. 31 A number of cellular factors have been demonstrated to negatively regulate Jak/STAT activities, including SHP-1 (SH2-homology-containing protein-tyrosine phosphatase-1), PIAS-3 (protein inhibitors of activated STATs), SOCS-1 (suppressors of cytokine signaling), and CIS (cytokine-in...