Regulatory dynamics of 11p13 suggest a role for EHF in modifying CF lung disease severity.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), but are not good predictors of lung phenotype. Genome-wide association studies (GWAS) previously identified additional genomic sites associated with CF lung disease severity. One of these, at chromosome 11p13, is an intergenic region between Ets homologous factor (EHF) and Apaf-1 interacting protein (APIP). Our goal was to determine the functional significance of this region, which being intergenic is probably regulatory. To identify cis-acting elements, we used DNase-seq and H3K4me1 and H3K27Ac ChIP-seq to map open and active chromatin respectively, in lung epithelial cells. Two elements showed strong enhancer activity for the promoters of EHF and the 5' adjacent gene E47 like ETS transcription factor 5 (ELF5) in reporter gene assays. No enhancers of the APIP promoter were found. Circular chromosome conformation capture (4C-seq) identified direct physical interactions of elements within 11p13. This confirmed the enhancer-promoter associations, identified additional interacting elements and defined topologically associating domain (TAD) boundaries, enriched for CCCTC-binding factor (CTCF). No strong interactions were observed with the APIP promoter, which lies outside the main TAD encompassing the GWAS signal. These results focus attention on the role of EHF in modifying CF lung disease severity.

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.

miR-1343 attenuates pathways of fibrosis by targeting the TGF-ß receptors.

Irreversible respiratory obstruction resulting from progressive airway damage, inflammation and fibrosis is a feature of several chronic respiratory diseases, including cystic fibrosis (CF), idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD). The cytokine transforming growth factor ß (TGF-ß) has a pivotal role in promoting lung fibrosis and is implicated in respiratory disease severity. In the present study, we show that a previously uncharacterized miRNA, miR-1343, reduces the expression of both TGF-ß receptor 1 and 2 by directly targeting their 3'-UTRs. After TGF-ß exposure, elevated intracellular miR-1343 significantly decreases levels of activated TGF-ß effector molecules, pSMAD2 (phosphorylated SMAD2) and pSMAD3 (phosphorylated SMAD3), when compared with a non-targeting control miRNA. As a result, the abundance of fibrotic markers is reduced, cell migration into a scratch wound impaired and epithelial-to-mesenchymal transition (EMT) repressed. Mature miR-1343 is readily detected in human neutrophils and HL-60 cells and is activated in response to stress in A549 lung epithelial cells. miR-1343 may have direct therapeutic applications in fibrotic lung disease.

Validation and psychometric properties of the German Capability for Suicide Questionnaire.

OBJECTIVE: Our study aimed at the validation of the newly developed German Capability for Suicide Questionnaire, the GCSQ. It is supposed to assess both fearlessness of death and pain tolerance, both facets of the acquired capability to commit suicide as postulated by the interpersonal theory of suicide. METHODS: This cross-sectional study was conducted on two clinical (n=424) and an online sample (n=532). Factor structure, convergent and discriminant validity, predictive validity as well as test-retest reliability were investigated. RESULTS: Two factors-"Fearlessness of Death" and "Pain Tolerance"-were derived. One item, the "perceived capability" item, assesses the subject's self-perception of acquired capability. Both subscales and the "perceived capability"-item demonstrate good construct validity and a high test-retest reliability. Fearlessness of death proves to be predictive for the occurrence of suicidal behaviors, whereas the importance of pain tolerance for suicidal behaviors was not confirmed. The subject's perception of his own capability is of high predictive value for both attempt status and suicidal behaviors. CONCLUSION: The GCSQ seems to be a useful measure of pain tolerance, fearlessness of death and the self-perception of acquired capability of suicide.

Metabolic Stress Drives Keratinocyte Defenses against Staphylococcus aureus Infection.

Human skin is commonly colonized and infected by Staphylococcus aureus. Exactly how these organisms are sensed by keratinocytes has not been clearly delineated. Using a combination of metabolic and transcriptomic methodologies, we found that S. aureus infection is sensed as a metabolic stress by the hypoxic keratinocytes. This induces HIF1α signaling, which promotes IL-1ß production and stimulates aerobic glycolysis to meet the metabolic requirements of infection. We demonstrate that staphylococci capable of glycolysis, including WT and agr mutants, readily induce HIF1α responses. In contrast, Δpyk glycolytic mutants fail to compete with keratinocytes for their metabolic needs. Suppression of glycolysis using 2-DG blocked keratinocyte production of IL-1ß in vitro and significantly exacerbated the S. aureus cutaneous infection in a murine model. Our data suggest that S. aureus impose a metabolic stress on keratinocytes that initiates signaling necessary to promote both glycolysis and the proinflammatory response to infection.

Large Morel-Lavallée lesion presenting as fungating mass with skin ulceration.

A Morel-Lavallée lesion, a type of soft tissue degloving injury that has also been referred to as a chronic expanding hematoma, is a relatively rare condition that usually develops following traumatic injury. Here, we present a case of a 60-year-old male with a Morel-Lavallée lesion diagnosed over 5 years after a traumatic injury of the hip. He presented with a large fungating mass and overlying skin ulceration, which was highly suspicious for sarcoma. However, lack of other systemic findings and constitutional complaints, as well as negative imaging studies, did not support a diagnosis of malignancy. This information, combined with the history of remote trauma to the affected area, instead led us to suspect the alternative diagnosis of a Morel-Lavallée lesion. The diagnosis was later confirmed by pathology showing a chronic expanding hematoma. To our knowledge, a Morel-Lavallée lesion presenting as a fungating mass has not been previously described.

Small Intestine Early Innate Immunity Response during Intestinal Colonization by Escherichia coli Depends on Its Extra-Intestinal Virulence Status.

Uropathogenic Escherichia coli (UPEC) strains live as commensals in the digestive tract of the host, but they can also initiate urinary tract infections. The aim of this work was to determine how a host detects the presence of a new UPEC strain in the digestive tract. Mice were orally challenged with UPEC strains 536 and CFT073, non-pathogenic strain K12 MG1655, and ΔPAI-536, an isogenic mutant of strain 536 lacking all 7 pathogenicity islands whose virulence is drastically attenuated. Intestinal colonization was measured, and cytokine expression was determined in various organs recovered from mice after oral challenge. UPEC strain 536 efficiently colonized the mouse digestive tract, and prior Enterobacteriaceae colonization was found to impact strain 536 colonization efficiency. An innate immune response, detected as the production of TNFα, IL-6 and IL-10 cytokines, was activated in the ileum 48 hours after oral challenge with strain 536, and returned to baseline within 8 days, without a drop in fecal pathogen load. Although inflammation was detected in the ileum, histology was normal at the time of cytokine peak. Comparison of cytokine secretion 48h after oral gavage with E. coli strain 536, CFT073, MG1655 or ΔPAI-536 showed that inflammation was more pronounced with UPECs than with non-pathogenic or attenuated strains. Pathogenicity islands also seemed to be involved in host detection, as IL-6 intestinal secretion was increased after administration of E. coli strain 536, but not after administration of ΔPAI-536. In conclusion, UPEC colonization of the mouse digestive tract activates acute phase inflammatory cytokine secretion but does not trigger any pathological changes, illustrating the opportunistic nature of UPECs. This digestive tract colonization model will be useful for studying the factors controlling the switch from commensalism to pathogenicity.

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.

Acquired Capability for Suicide, Pain Tolerance, and Fearlessness of Pain-Validation of the Pain Tolerance Scale of the German Capability for Suicide Questionnaire.

The interpersonal theory of suicide (Joiner, 2005) postulates that for a serious suicide attempt, one has to possess the acquired capability to commit suicide. Acquired capability includes higher pain tolerance, which is further assumed to comprise both an elevated physical pain tolerance and fearlessness of pain. Recently, the German Capability for Suicide Questionnaire (GCSQ) was validated. The aim of this study is further validation of the GCSQ's Pain Tolerance scale by investigating the scale's association with objective pain tolerance and fearlessness of pain in two undergraduate samples (N = 81; N = 76). Both associations were found indicating a strong criterion validity of the Pain Tolerance scale.

The german version of the painful and provocative events scale: a psychometric investigation.

The interpersonal theory of suicide (Joiner, T.E., 2005. Why People Die By Suicide. Harvard University Press, Cambridge) postulates that, for a serious or lethal suicide attempt one has to possess a desire to die and the capability to commit suicide. The capability is proposed to be acquired over time by repeated experiences with painful and provocative events such as self-injurious behavior and other experiences such as childhood abuse, combat exposure, physical fights, promiscuous sex, and playing contact sports. Up to now, experiences with painful and provocative events are measured with various versions of the Painful and Provocative Events Scale (PPES). However, a thorough validation of this assessment instrument is still lacking. Our study aimed at validating the German version of PPES, with two clinical (n=424) and one community sample (n=532). Results support a two-factor structure (eight items "active painful and provocative events", four items "passive painful and provocative events") that was invariant across the three subsamples. Nonetheless, low factor loadings, low indicator reliabilities, moderate construct reliability and mixed evidence for construct validity indicate that the PPES in its current form appears to be of limited use. The development of a new instrument to assess painful and provocative events seems appropriate.

Methicillin-resistant Staphylococcus aureus adaptation to human keratinocytes.

UNLABELLED: Skin is the most common site of Staphylococcus aureus infection. While most of these infections are self-limited, recurrent infections are common. Keratinocytes and recruited immune cells participate in skin defense against infection. We postulated that S. aureus is able to adapt to the milieu within human keratinocytes to avoid keratinocyte-mediated clearance. From a collection of S. aureus isolated from chronically infected patients with atopic dermatitis, we noted 22% had an agr mutant-like phenotype. Using several models of human skin infection, we demonstrate that toxin-deficient, agr mutants of methicillin-resistant S. aureus (MRSA) USA300 are able to persist within keratinocytes by stimulating autophagy and evading caspase-1 and inflammasome activation. MRSA infection induced keratinocyte autophagy, as evidenced by galectin-8 and LC3 accumulation. Autophagy promoted the degradation of inflammasome components and facilitated staphylococcal survival. The recovery of more than 58% agr or RNAIII mutants (P < 0.0001) of an inoculum of wild-type (WT) MRSA from within wortmannin-treated keratinocytes compared to control keratinocytes reflected the survival advantage for mutants no longer expressing agr-dependent toxins. Our results illustrate the dynamic interplay between S. aureus and keratinocytes that can result in the selection of mutants that have adapted specifically to evade keratinocyte-mediated clearance mechanisms. IMPORTANCE: Human skin is a major site of staphylococcal infection, and keratinocytes actively participate in eradication of these pathogens. We demonstrate that methicillin-resistant Staphylococcus aureus (MRSA) is ingested by keratinocytes and activates caspase-1-mediated clearance through pyroptosis. Toxin-deficient MRSA mutants are selected within keratinocytes that fail to induce caspase-1 activity and keratinocyte-mediated clearance. These intracellular staphylococci induce autophagy that enhances their intracellular survival by diminishing inflammasome components. These findings suggest that S. aureus mutants, by exploiting autophagy, can persist within human keratinocytes.