Cystic Fibrosis Transmembrane Conductance Regulator Attaches Tumor Suppressor PTEN to the Membrane and Promotes Anti Pseudomonas aeruginosa Immunity.

The tumor suppressor PTEN controls cell proliferation by regulating phosphatidylinositol-3-kinase (PI3K) activity, but the participation of PTEN in host defense against bacterial infection is less well understood. Anti-inflammatory PI3K-Akt signaling is suppressed in patients with cystic fibrosis (CF), a disease characterized by hyper-inflammatory responses to airway infection. We found that Ptenl-/- mice, which lack the NH2-amino terminal splice variant of PTEN, were unable to eradicate Pseudomonas aeruginosa from the airways and could not generate sufficient anti-inflammatory PI3K activity, similar to what is observed in CF. PTEN and the CF transmembrane conductance regulator (CFTR) interacted directly and this interaction was necessary to position PTEN at the membrane. CF patients under corrector-potentiator therapy, which enhances CFTR transport to the membrane, have increased PTEN amounts. These findings suggest that improved CFTR trafficking could enhance P. aeruginosa clearance from the CF airway by activating PTEN-mediated anti-bacterial responses and might represent a therapeutic strategy.

Humanized Mice Exhibit Increased Susceptibility to Staphylococcus aureus Pneumonia.

Staphylococcus aureus is a highly successful human pathogen that has evolved in response to human immune pressure. The common USA300 methicillin-resistant S. aureus (MRSA) strains express a number of toxins, such as Panton-Valentine leukocidin and LukAB, that have specificity for human receptors. Using nonobese diabetic (NOD)-scid IL2Rγnull (NSG) mice reconstituted with a human hematopoietic system, we were able to discriminate the roles of these toxins in the pathogenesis of pneumonia. We demonstrate that expression of human immune cells confers increased severity of USA300 infection. The expression of PVL but not LukAB resulted in more-severe pulmonary infection by the wild-type strain (with a 30-fold increase in the number of colony-forming units/mL; P < .01) as compared to infection with the lukS/F-PV (Δpvl) mutant. Treatment of mice with anti-PVL antibody also enhanced bacterial clearance. We found significantly greater numbers (by 95%; P < .05) of macrophages in the airways of mice infected with the Δpvl mutant compared with those infected with the wild-type strain, as well as significantly greater expression of human tumor necrosis factor and interleukin 6 (84% and 51% respectively; P < .01). These results suggest that the development of humanized mice may provide a framework to assess the contribution of human-specific toxins and better explore the roles of specific components of the human immune system in protection from S. aureus infection.

Toxin-induced necroptosis is a major mechanism of Staphylococcus aureus lung damage.

Staphylococcus aureus USA300 strains cause a highly inflammatory necrotizing pneumonia. The virulence of this strain has been attributed to its expression of multiple toxins that have diverse targets including ADAM10, NLRP3 and CD11b. We demonstrate that induction of necroptosis through RIP1/RIP3/MLKL signaling is a major consequence of S. aureus toxin production. Cytotoxicity could be prevented by inhibiting either RIP1 or MLKL signaling and S. aureus mutants lacking agr, hla or Hla pore formation, lukAB or psms were deficient in inducing cell death in human and murine immune cells. Toxin-associated pore formation was essential, as cell death was blocked by exogenous K+ or dextran. MLKL inhibition also blocked caspase-1 and IL-1ß production, suggesting a link to the inflammasome. Rip3(-/-) mice exhibited significantly improved staphylococcal clearance and retained an alveolar macrophage population with CD200R and CD206 markers in the setting of acute infection, suggesting increased susceptibility of these leukocytes to necroptosis. The importance of this anti-inflammatory signaling was indicated by the correlation between improved outcome and significantly decreased expression of KC, IL-6, TNF, IL-1α and IL-1ß in infected mice. These findings indicate that toxin-induced necroptosis is a major cause of lung pathology in S. aureus pneumonia and suggest the possibility of targeting components of this signaling pathway as a therapeutic strategy.

A TLR and non-TLR mediated innate response to lentiviruses restricts hepatocyte entry and can be ameliorated by pharmacological blockade.

Lentiviral vector (LV)-mediated gene transfer is a promising method of gene therapy. We previously reported that systemic injection of HIV-based LV triggers a transient inflammatory response. Here, we carried out studies to better characterize this response, and to develop a strategy to overcome the adverse effects of interferon (IFN) on LV-mediated gene transfer. We profiled gene expression in the liver after LV administration using deep-sequencing (RNA-seq), and identified several innate response pathways. We examined the response to LV in MyD88-TRIF knockout mice, which are incapable of toll-like receptor (TLR) signaling. Unexpectedly, the IFN response to LV was not reduced in the liver indicating that a non-TLR pathway can recognize LV in this organ. Indeed, blocking reverse transcription with azidothymidine (AZT) reduced the IFN response only in the liver, suggesting that proviral DNA can be a trigger. To block the inflammatory response, we pretreated mice with a short course of dexamethasone (Dex). At 4 hours post-treatment, all the IFN-induced genes were normalized. By blocking the inflammatory response, hepatocyte transduction was dramatically increased, which in turn doubled the level of human factor IX (FIX) produced by a hepatocyte-specific LV. Our studies uncover new insights into LV-induced immune responses in the liver, and provide a means to increase the safety and efficiency of LV-mediated gene transfer.

Necroptosis Promotes Staphylococcus aureus Clearance by Inhibiting Excessive Inflammatory Signaling.

Staphylococcus aureus triggers inflammation through inflammasome activation and recruitment of neutrophils, responses that are critical for pathogen clearance but are associated with substantial tissue damage. We postulated that necroptosis, cell death mediated by the RIPK1/RIPK3/MLKL pathway, would function to limit pathological inflammation. In models of skin infection or sepsis, Mlkl-/- mice had high bacterial loads, an inability to limit interleukin-1b (IL-1b) production, and excessive inflammation. Similarly, mice treated with RIPK1 or RIPK3 inhibitors had increased bacterial loads in a model of sepsis. Ripk3-/- mice exhibited increased staphylococcal clearance and decreased inflammation in skin and systemic infection, due to direct effects of RIPK3 on IL-1b activation and apoptosis. In contrast to Casp1/4-/- mice with defective S. aureus killing, the poor outcomes of Mlkl-/- mice could not be attributed to impaired phagocytic function. We conclude that necroptotic cell death limits the pathological inflammation induced by S. aureus.