The ENaC-overexpressing mouse as a model of cystic fibrosis lung disease

AbstractChronic lung disease remains the major cause of morbidity and mortality of cystic fibrosis (CF) patients. Cftr mutant mice developed severe intestinal obstruction, but did not exhibit the characteristic CF ion transport defects (i.e. deficient cAMP-dependent Cl− secretion and increased Na+ absorption) in the lower airways, and failed to develop CF-like lung disease. These observations led to the generation of transgenic mice with airway-specific overexpression of the epithelial Na+ channel (ENaC) as an alternative approach to mimic CF ion transport pathophysiology in the lung. Studies of the phenotype of βENaC-transgenic mice demonstrated that increased airway Na+ absorption causes airway surface liquid (ASL) depletion, reduced mucus transport and a spontaneous CF-like lung disease with airway mucus obstruction and chronic airway inflammation. Here, we summarize approaches that can be applied for studies of the complex in vivo pathogenesis and preclinical evaluation of novel therapeutic strategies in this model of CF lung disease

Increased nuclear suppressor of cytokine signaling 1 in asthmatic bronchial epithelium suppresses rhinovirus induction of innate interferons

Background Rhinovirus infections are the dominant cause of asthma exacerbations, and deficient virus induction of IFN-α/β/λ in asthmatic patients is important in asthma exacerbation pathogenesis. Mechanisms causing this interferon deficiency in asthmatic patients are unknown. Objective We sought to investigate the expression of suppressor of cytokine signaling (SOCS) 1 in tissues from asthmatic patients and its possible role in impaired virus-induced interferon induction in these patients. Methods We assessed SOCS1 mRNA and protein levels in vitro, bronchial biopsy specimens, and mice. The role of SOCS1 was inferred by proof-of-concept studies using overexpression with reporter genes and SOCS1-deficient mice. A nuclear role of SOCS1 was shown by using bronchial biopsy staining, overexpression of mutant SOCS1 constructs, and confocal microscopy. SOCS1 levels were also correlated with asthma-related clinical outcomes. Results We report induction of SOCS1 in bronchial epithelial cells (BECs) by asthma exacerbation–related cytokines and by rhinovirus infection in vitro. We found that SOCS1 was increased in vivo in bronchial epithelium and related to asthma severity. SOCS1 expression was also increased in primary BECs from asthmatic patients ex vivo and was related to interferon deficiency and increased viral replication. In primary human epithelium, mouse lung macrophages, and SOCS1-deficient mice, SOCS1 suppressed rhinovirus induction of interferons. Suppression of virus-induced interferon levels was dependent on SOCS1 nuclear translocation but independent of proteasomal degradation of transcription factors. Nuclear SOCS1 levels were also increased in BECs from asthmatic patients. Conclusion We describe a novel mechanism explaining interferon deficiency in asthmatic patients through a novel nuclear function of SOCS1 and identify SOCS1 as an important therapeutic target for asthma exacerbations

In Search of Patient Zero: Visual Analytics of Pathogen Transmission Pathways in Hospitals

Pathogen outbreaks (i.e., outbreaks of bacteria and viruses) in hospitals can cause high mortality rates and increase costs for hospitals significantly. An outbreak is generally noticed when the number of infected patients rises above an endemic level or the usual prevalence of a pathogen in a defined population. Reconstructing transmission pathways back to the source of an outbreak -- the patient zero or index patient -- requires the analysis of microbiological data and patient contacts. This is often manually completed by infection control experts. We present a novel visual analytics approach to support the analysis of transmission pathways, patient contacts, the progression of the outbreak, and patient timelines during hospitalization. Infection control experts applied our solution to a real outbreak of Klebsiella pneumoniae in a large German hospital. Using our system, our experts were able to scale the analysis of transmission pathways to longer time intervals (i.e., several years of data instead of days) and across a larger number of wards. Also, the system is able to reduce the analysis time from days to hours. In our final study, feedback from twenty-five experts from seven German hospitals provides evidence that our solution brings significant benefits for analyzing outbreaks

Crucial Role of Nucleic Acid Sensing via Endosomal Toll-Like Receptors for the Defense of Streptococcus pyogenes in vitro and in vivo

Streptococcus pyogenes is a major human pathogen causing a variety of diseases ranging from common pharyngitis to life-threatening soft tissue infections and sepsis. Microbial nucleic acids, especially bacterial RNA, have recently been recognized as a major group of pathogen-associated molecular patterns (PAMPs) involved in the detection of Streptococcus pyogenes via endosomal Toll-like receptors (TLRs) in vitro. However, the individual contribution and cooperation between TLRs as well as cell-type and strain specific differences in dependency on nucleic acid detection during S. pyogenes infection in vitro have not been clarified in detail. Moreover, the role of particularly bacterial RNA for the defense of S. pyogenes infection in vivo remains poorly defined. In this study, we report that in all investigated innate immune cells involved in the resolution of bacterial infections, including murine macrophages, dendritic cells and neutrophils, recognition of S. pyogenes strain ATCC12344 is almost completely dependent on nucleic acid sensing via endosomal TLRs at lower MOIs, whereas at higher MOIs, detection via TLR2 plays an additional, yet redundant role. We further demonstrate that different S. pyogenes strains display a considerable inter-strain variability with respect to their nucleic acid dependent recognition. Moreover, TLR13-dependent recognition of S. pyogenes RNA is largely non-redundant in bone marrow-derived macrophages (BMDMs), but less relevant in neutrophils and bone marrow-derived myeloid dendritic cells (BMDCs) for the induction of an innate immune response in vitro. In vivo, we show that a loss of nucleic acid sensing blunts early recognition of S. pyogenes, leading to a reduced local containment of the bacterial infection with subsequent pronounced systemic inflammation at later time points. Thus, our results argue for a crucial role of nucleic acid sensing via endosomal TLRs in defense of S. pyogenes infection both in vitro and in vivo

A mouse model of post-stroke pneumonia induced by intra-tracheal inoculation with Streptococcus pneumoniae

Background: Stroke-induced immunodeficiency increases the risk of infectious complications, which adversely affects neurological outcome. Among those, pneumonia affects as many as one third of stroke patients and is the main contributor to mortality in the post-acute phase of stroke. Experimental findings on post-stroke susceptibility to spontaneous pneumonia in mice are contradictory. Here, we established a mouse model inducing standardized bacterial pneumonia and characterized the impaired pulmonary cellular and humoral immune responses after experimental stroke. Methods: Bacterial pneumonia was induced by intra-tracheal inoculation with Streptococcus pneumoniae at different time points after transient middle cerebral artery occlusion (MCAO). Bacterial counts in lungs and blood, histological changes, and cytokine production in the lungs were assessed. Furthermore, we investigated the effect of pneumonia on stroke outcome. Results: Intra-tracheal inoculation resulted in reproducible pneumonia and bacteraemia, and demonstrated post-stroke susceptibility to streptococcal pneumonia developing with a delay of at least 24 h after MCAO. Higher bacterial counts in mice infected 3 days after stroke induction correlated with reduced neutrophil and macrophage infiltration in the lungs and lower levels of pro-inflammatory cytokines in the broncho-alveolar lavage compared to sham-operated animals. Pneumonia increased mortality without affecting brain-infiltrating leukocytes. Conclusions: In this standardized mouse model of post-stroke pneumonia, we describe attenuated leukocyte infiltration and cytokine production in response to bacterial infection in the lungs that has a profound effect on outcome

Gut microbiome patterns correlate with higher postoperative complication rates after pancreatic surgery

Abstract Background Postoperative complications are of great relevance in daily clinical practice, and the gut microbiome might play an important role by preventing pathogens from crossing the intestinal barrier. The two aims of this prospective clinical pilot study were: (1) to examine changes in the gut microbiome following pancreatic surgery, and (2) to correlate these changes with the postoperative course of the patient. Results In total, 116 stool samples of 32 patients undergoing pancreatic surgery were analysed by 16S-rRNA gene next-generation sequencing. One sample per patient was collected preoperatively in order to determine the baseline gut microbiome without exposure to surgical stress and/or antibiotic use. At least two further samples were obtained within the first 10 days following the surgical procedure to observe longitudinal changes in the gut microbiome. Whenever complications occurred, further samples were examined. Based on the structure of the gut microbiome, the samples could be allocated into three different microbial communities (A, B and C). Community B showed an increase in Akkermansia, Enterobacteriaceae and Bacteroidales as well as a decrease in Lachnospiraceae, Prevotella and Bacteroides. Patients showing a microbial composition resembling community B at least once during the observation period were found to have a significantly higher risk for developing postoperative complications (B vs. A, odds ratio = 4.96, p < 0.01**; B vs. C, odds ratio = 2.89, p = 0.019*). Conclusions The structure of the gut microbiome is associated with the development of postoperative complications

Toll-Like Receptor 3 and Suppressor of Cytokine Signaling Proteins Regulate CXCR4 and CXCR7 Expression in Bone Marrow-Derived Human Multipotent Stromal Cells

The use of bone marrow-derived human multipotent stromal cells (hMSC) in cell-based therapies has dramatically increased in recent years, as researchers have exploited the ability of these cells to migrate to sites of tissue injury, inflammation, and tumors. Our group established that hMSC respond to "danger" signals--by-products of damaged, infected or inflamed tissues--via activation of Toll-like receptors (TLRs). However, little is known regarding downstream signaling mediated by TLRs in hMSC.We demonstrate that TLR3 stimulation activates a Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 1 pathway, and increases expression of suppressor of cytokine signaling (SOCS) 1 and SOCS3 in hMSC. Our studies suggest that each of these SOCS plays a distinct role in negatively regulating TLR3 and JAK/STAT signaling. TLR3-mediated interferon regulatory factor 1 (IRF1) expression was inhibited by SOCS3 overexpression in hMSC while SOCS1 overexpression reduced STAT1 activation. Furthermore, our study is the first to demonstrate that when TLR3 is activated in hMSC, expression of CXCR4 and CXCR7 is downregulated. SOCS3 overexpression inhibited internalization of both CXCR4 and CXCR7 following TLR3 stimulation. In contrast, SOCS1 overexpression only inhibited CXCR7 internalization.These results demonstrate that SOCS1 and SOCS3 each play a functionally distinct role in modulating TLR3, JAK/STAT, and CXCR4/CXCR7 signaling in hMSC and shed further light on the way hMSC respond to danger signals