Quorum-sensing agr system of Staphylococcus aureus primes gene expression for protection from lethal oxidative stress.

The agr quorum-sensing system links Staphylococcus aureus metabolism to virulence, in part by increasing bacterial survival during exposure to lethal concentrations of H2O2, a crucial host defense against S. aureus. We now report that protection by agr surprisingly extends beyond post-exponential growth to the exit from stationary phase when the agr system is no longer turned on. Thus, agr can be considered a constitutive protective factor. Deletion of agr resulted in decreased ATP levels and growth, despite increased rates of respiration or fermentation at appropriate oxygen tensions, suggesting that Δagr cells undergo a shift towards a hyperactive metabolic state in response to diminished metabolic efficiency. As expected from increased respiratory gene expression, reactive oxygen species (ROS) accumulated more in the agr mutant than in wild-type cells, thereby explaining elevated susceptibility of Δagr strains to lethal H2O2 doses. Increased survival of wild-type agr cells during H2O2 exposure required sodA, which detoxifies superoxide. Additionally, pretreatment of S. aureus with respiration-reducing menadione protected Δagr cells from killing by H2O2. Thus, genetic deletion and pharmacologic experiments indicate that agr helps control endogenous ROS, thereby providing resilience against exogenous ROS. The long-lived 'memory' of agr-mediated protection, which is uncoupled from agr activation kinetics, increased hematogenous dissemination to certain tissues during sepsis in ROS-producing, wild-type mice but not ROS-deficient (Cybb-/-) mice. These results demonstrate the importance of protection that anticipates impending ROS-mediated immune attack. The ubiquity of quorum sensing suggests that it protects many bacterial species from oxidative damage.

Quorum-sensing agr system of Staphylococcus aureus primes gene expression for protection from lethal oxidative stress.

The agr quorum-sensing system links Staphylococcus aureus metabolism to virulence, in part by increasing bacterial survival during exposure to lethal concentrations of H2O2, a crucial host defense against S. aureus. We now report that protection by agr surprisingly extends beyond post-exponential growth to the exit from stationary phase when the agr system is no longer turned on. Thus, agr can be considered a constitutive protective factor. Deletion of agr increased both respiration and fermentation but decreased ATP levels and growth, suggesting that Δagr cells assume a hyperactive metabolic state in response to reduced metabolic efficiency. As expected from increased respiratory gene expression, reactive oxygen species (ROS) accumulated more in the agr mutant than in wild-type cells, thereby explaining elevated susceptibility of Δagr strains to lethal H2O2 doses. Increased survival of wild-type agr cells during H2O2 exposure required sodA, which detoxifies superoxide. Additionally, pretreatment of S. aureus with respiration-reducing menadione protected Δagr cells from killing by H2O2. Thus, genetic deletion and pharmacologic experiments indicate that agr helps control endogenous ROS, thereby providing resilience against exogenous ROS. The long-lived "memory" of agr-mediated protection, which is uncoupled from agr activation kinetics, increased hematogenous dissemination to certain tissues during sepsis in ROS-producing, wild-type mice but not ROS-deficient (Nox2-/-) mice. These results demonstrate the importance of protection that anticipates impending ROS-mediated immune attack. The ubiquity of quorum sensing suggests that it protects many bacterial species from oxidative damage.

Improvement in postoperative lung function in patients with moderate to severe airway obstruction after robotic-assisted thoracoscopic tracheobronchoplasty.

OBJECTIVE: The study objective was to examine pulmonary function and quality of life improvement after robotic-assisted thoracoscopic tracheobronchoplasty for patients with different degrees of obstructive airway disease. METHODS: We performed a retrospective review of a prospective database of patients who underwent robotic-assisted thoracoscopic tracheobronchoplasty between 2013 and 2020. RESULTS: A total of 118 patients underwent robotic-assisted thoracoscopic tracheobronchoplasty. Preoperative and postoperative pulmonary function tests were available for 108 patients. Postoperative pulmonary function tests at a median of 16 months demonstrated a significant increase in percent predicted forced expiratory volume in 1 second (preoperative median: 76.76% predicted, postoperative: 83% predicted, P = .002). Preoperative and postoperative St George Respiratory Questionnaires were available for 64 patients with a significant decrease in postoperative score at a median of 7 months (preoperative median: 61, postoperative: 41.60, P < .001). When stratified by preoperative degree of obstruction, robotic-assisted thoracoscopic tracheobronchoplasty improved forced expiratory volume in 1 second in moderate to very severe obstruction with a statistically significant improvement in moderate (preoperative median: 63.91% predicted, postoperative median: 73% predicted, P = .001) and severe (preoperative median: 44% predicted, postoperative median: 57% predicted, P = .007) obstruction. St George Respiratory Questionnaire scores improved for all patients. Improvement for mild (preoperative median: 61.27, postoperative median: 36.71, P < .001) and moderate (preoperative median: 57.15, postoperative median: 47.52, P = .03) obstruction was statistically significant. CONCLUSIONS: Robotic-assisted thoracoscopic tracheobronchoplasty improves obstruction and symptoms. With limited follow-up, subgroup analysis showed forced expiratory volume in 1 second improved in severe preoperative obstruction and quality of life improved in moderate obstruction. Future follow-up is required to determine robotic-assisted thoracoscopic tracheobronchoplasty effects on the most severe group, but we cannot conclude that increased degree of preoperative obstruction precludes surgery.

Robotic-assisted tracheobronchoplasty: Quality of life and pulmonary function assessment on intermediate follow-up.

OBJECTIVE: The initial description of robotic tracheobronchoplasty for the treatment of tracheobronchomalacia demonstrated feasibility, safety, and short-term symptomatic and functional improvement. The purpose of the current study was to demonstrate intermediate outcomes in postoperative pulmonary function and quality of life after robotic tracheobronchoplasty. METHODS: We retrospectively reviewed prospectively collected clinical data from 42 patients who underwent robotic tracheobronchoplasty from May 2016 to December 2017. The Institutional Review Board or equivalent ethics committee of the Northwell Health approved the study protocol and publication of data. Patient written consent for the publication of the study data was waived by the Institutional Review Board. RESULTS: A total of 42 patients underwent robotic tracheobronchoplasty during the study period. Median total follow-up is 40 months. There was 1 death since surgery from an unrelated disease. Significant decreases in St George's Respiratory Questionnaire total score (preoperative mean: 64.01, postoperative mean: 38.91, P = .002), St George's Respiratory Questionnaire symptom score (preoperative median: 82.6, postoperative median: 43.99, P < .001), and St George's Respiratory Questionnaire impact score (preoperative median: 55.78, postoperative median: 25.95, P < .001) were apparent at a median follow-up of 13 months. Comparison of preoperative and postoperative pulmonary function tests revealed a significant increase in percent predicted forced expiratory volume in 1 second (preoperative median: 74% vs postoperative median: 82%, P = .001), forced vital capacity (preoperative median: 68.5% vs postoperative median: 80.63%, P < .001), and peak expiratory flow (preoperative median: 61.5% vs postoperative median: 75%, P = .02) measured at a median follow-up of 29 months. CONCLUSIONS: Robotic tracheobronchoplasty is associated with low intermediate-term mortality. Robotic tracheobronchoplasty results in significant improvement in quality of life and postoperative pulmonary function. Longer-term follow-up is necessary to continue to elucidate the effect of robotic tracheobronchoplasty on halting pathologic progression of tracheobronchomalacia and to determine the long-term impact of tracheobronchoplasty on symptomatic and functional improvement.

Lung CD4+ resident memory T cells remodel epithelial responses to accelerate neutrophil recruitment during pneumonia.

Previous pneumococcal experience establishes lung-resident IL-17A-producing CD4+ memory TRM cells that accelerate neutrophil recruitment against heterotypic pneumococci. Herein, we unravel a novel crosstalk between CD4+ TRM cells and lung epithelial cells underlying this protective immunity. Depletion of CD4+ cells in pneumococcus-experienced mice diminished CXCL5 (but not CXCL1 or CXCL2) and downstream neutrophil accumulation in the lungs. Epithelial cells from experienced lungs exhibited elevated mRNA for CXCL5 but not other epithelial products such as GM-CSF or CCL20, suggesting a skewing by CD4+ TRM cells. Genome-wide expression analyses revealed a significant remodeling of the epithelial transcriptome of infected lungs due to infection history, ~80% of which was CD4+ cell-dependent. The CD4+ TRM cell product IL-17A stabilized CXCL5 but not GM-CSF or CCL20 mRNA in cultured lung epithelial cells, implicating posttranscriptional regulation as a mechanism for altered epithelial responses. These results suggest that epithelial cells in experienced lungs are effectively different, owing to their communication with TRM cells. Our study highlights the role of tissue-resident adaptive immune cells in fine-tuning epithelial functions to hasten innate immune responses and optimize defense in experienced lungs, a concept that may apply broadly to mucosal immunology.

Minimally invasive robotic-assisted excision of an aortopulmonary mediastinal paraganglioma.

Aortopulmonary paragangliomas are rare, neural crest-derived tumours that arise in the aortopulmonary window and present a significant surgical challenge due to their proximity to major vessels. In the relatively few cases reported in the study, thoracotomy and video-assisted thorascopic surgery (VATS) approaches have been utilized for the treatment of such masses. In this report, we describe the application of a totally endoscopic robotic-assisted VATS approach for the excision of an aortopulmonary paraganglioma.

MicroRNA Signature of Cigarette Smoking and Evidence for a Putative Causal Role of MicroRNAs in Smoking-Related Inflammation and Target Organ Damage.

BACKGROUND: Cigarette smoking increases risk for multiple diseases. MicroRNAs regulate gene expression and may play a role in smoking-induced target organ damage. We sought to describe a microRNA signature of cigarette smoking and relate it to smoking-associated clinical phenotypes, gene expression, and lung inflammatory signaling. METHODS AND RESULTS: Expression profiling of 283 microRNAs was conducted on whole blood-derived RNA from 5023 Framingham Heart Study participants (54.0% women; mean age, 55±13 years) using TaqMan assays and high-throughput reverse transcription quantitative polymerase chain reaction. Associations of microRNA expression with smoking status and associations of smoking-related microRNAs with inflammatory biomarkers and pulmonary function were tested with linear mixed effects models. We identified a 6-microRNA signature of smoking. Five of the 6 smoking-related microRNAs were associated with serum levels of C-reactive protein or interleukin-6; miR-1180 was associated with pulmonary function measures at a marginally significant level. Bioinformatic evaluation of smoking-associated genes coexpressed with the microRNA signature of cigarette smoking revealed enrichment for immune-related pathways. Smoking-associated microRNAs altered expression of selected inflammatory mediators in cell culture gain-of-function assays. CONCLUSIONS: We characterized a novel microRNA signature of cigarette smoking. The top microRNAs were associated with systemic inflammatory markers and reduced pulmonary function, correlated with expression of genes involved in immune function, and were sufficient to modulate inflammatory signaling. Our results highlight smoking-associated microRNAs and are consistent with the hypothesis that smoking-associated microRNAs serve as mediators of smoking-induced inflammation and target organ damage. These findings call for further mechanistic studies to explore the diagnostic and therapeutic use of smoking-related microRNAs.

Expression of Piwi protein MIWI2 defines a distinct population of multiciliated cells.

P-element-induced wimpy testes (Piwi) proteins are known for suppressing retrotransposon activation in the mammalian germline. However, whether Piwi protein or Piwi-dependent functions occur in the mammalian soma is unclear. Contrary to germline-restricted expression, we observed that Piwi-like Miwi2 mRNA is indeed expressed in epithelial cells of the lung in adult mice and that it is induced during pneumonia. Further investigation revealed that MIWI2 protein localized to the cytoplasm of a discrete population of multiciliated airway epithelial cells. Isolation and next-generation sequencing of MIWI2-positive multiciliated cells revealed that they are phenotypically distinct from neighboring MIWI2-negative multiciliated cells. Mice lacking MIWI2 exhibited an altered balance of airway epithelial cells, demonstrating fewer multiciliated cells and an increase in club cells. During pneumococcal pneumonia, Miwi2-deficient mice exhibited increased expression of inflammatory mediators and increased immune cell recruitment, leading to enhanced bacterial clearance. Taken together, our data delineate MIWI2-dependent functions outside of the germline and demonstrate the presence of distinct subsets of airway multiciliated cells that can be discriminated by MIWI2 expression. By demonstrating roles for MIWI2 in airway cell identity and pulmonary innate immunity, these studies elucidate unanticipated physiological functions for Piwi proteins in somatic tissues.

The RNA uridyltransferase Zcchc6 is expressed in macrophages and impacts innate immune responses.

Alveolar macrophages orchestrate pulmonary innate immunity and are essential for early immune surveillance and clearance of microorganisms in the airways. Inflammatory signaling must be sufficiently robust to promote host defense but limited enough to prevent excessive tissue injury. Macrophages in the lungs utilize multiple transcriptional and post-transcriptional mechanisms of inflammatory gene expression to delicately balance the elaboration of immune mediators. RNA terminal uridyltransferases (TUTs), including the closely homologous family members Zcchc6 (TUT7) and Zcchc11 (TUT4), have been implicated in the post-transcriptional regulation of inflammation from studies conducted in vitro. In vivo, we observed that Zcchc6 is expressed in mouse and human primary macrophages. Zcchc6-deficient mice are viable and born in Mendelian ratios and do not exhibit an observable spontaneous phenotype under basal conditions. Following an intratracheal challenge with S. pneumoniae, Zcchc6 deficiency led to a modest but significant increase in the expression of select cytokines including IL-6, CXCL1, and CXCL5. These findings were recapitulated in vitro whereby Zcchc6-deficient macrophages exhibited similar increases in cytokine expression due to bacterial stimulation. Although loss of Zcchc6 also led to increased neutrophil emigration to the airways during pneumonia, these responses were not sufficient to impact host defense against infection.

Myeloid-epithelial cross talk coordinates synthesis of the tissue-protective cytokine leukemia inhibitory factor during pneumonia.

In bacterial pneumonia, lung damage resulting from epithelial cell injury is a major contributor to the severity of disease and, in some cases, can lead to long-term sequelae, especially in the setting of severe lung injury or acute respiratory distress syndrome. Leukemia inhibitory factor (LIF), a member of the IL-6 cytokine family, is a critical determinant of lung tissue protection during pneumonia, but the cellular sources of LIF and the signaling pathways leading to its production in the infected lung are not known. Here, we demonstrate that lung epithelium, specifically alveolar type II cells, is the predominant site of LIF transcript induction in pneumonic mouse lungs. Epithelial cell cultures were induced to express LIF by bacteria and by sterile bronchoalveolar lavage fluid from pneumonic mice. Reciprocal bone marrow chimera studies demonstrated that LIF deficiency in the nonhematopoietic compartment, but not LIF deficiency in hematopoietic cells, eliminated LIF induction during pneumonia. Although NF-κB RelA (p65) is essential for the expression of many cytokines during pneumonia, its targeted mutation in the lung epithelium was inconsequential for pneumonia-driven LIF induction. However, maximal expression of this epithelial-derived cytokine was dependent on NF-κB RelA in myeloid cells. Overall, our data suggest a signaling axis whereby activation of NF-κB RelA in myeloid cells promotes epithelial LIF induction during lung infections, representing a means through which these two cell types collaborate to improve tissue resilience during pneumonia.

Epithelial Cell-Derived Secreted and Transmembrane 1a Signals to Activated Neutrophils during Pneumococcal Pneumonia.

Airway epithelial cell responses are critical to the outcome of lung infection. In this study, we aimed to identify unique contributions of epithelial cells during lung infection. To differentiate genes induced selectively in epithelial cells during pneumonia, we compared genome-wide expression profiles from three sorted cell populations: epithelial cells from uninfected mouse lungs, epithelial cells from mouse lungs with pneumococcal pneumonia, and nonepithelial cells from those same infected lungs. Of 1,166 transcripts that were more abundant in epithelial cells from infected lungs compared with nonepithelial cells from the same lungs or from epithelial cells of uninfected lungs, 32 genes were identified as highly expressed secreted products. Especially strong signals included two related secreted and transmembrane (Sectm) 1 genes, Sectm1a and Sectm1b. Refinement of sorting strategies suggested that both Sectm1 products were induced predominantly in conducting airway epithelial cells. Sectm1 was induced during the early stages of pneumococcal pneumonia, and mutation of NF-κB RelA in epithelial cells did not diminish its expression. Instead, type I IFN signaling was necessary and sufficient for Sectm1 induction in lung epithelial cells, mediated by signal transducer and activator of transcription 1. For target cells, Sectm1a bound to myeloid cells preferentially, in particular Ly6G(bright)CD11b(bright) neutrophils in the infected lung. In contrast, Sectm1a did not bind to neutrophils from uninfected lungs. Sectm1a increased expression of the neutrophil-attracting chemokine CXCL2 by neutrophils from the infected lung. We propose that Sectm1a is an epithelial product that sustains a positive feedback loop amplifying neutrophilic inflammation during pneumococcal pneumonia.

Bacterial Hypoxic Responses Revealed as Critical Determinants of the Host-Pathogen Outcome by TnSeq Analysis of Staphylococcus aureus Invasive Infection.

Staphylococcus aureus is capable of infecting nearly every organ in the human body. In order to infiltrate and thrive in such diverse host tissues, staphylococci must possess remarkable flexibility in both metabolic and virulence programs. To investigate the genetic requirements for bacterial survival during invasive infection, we performed a transposon sequencing (TnSeq) analysis of S. aureus during experimental osteomyelitis. TnSeq identified 65 genes essential for staphylococcal survival in infected bone and an additional 148 mutants with compromised fitness in vivo. Among the loci essential for in vivo survival was SrrAB, a staphylococcal two-component system previously reported to coordinate hypoxic and nitrosative stress responses in vitro. Healthy bone is intrinsically hypoxic, and intravital oxygen monitoring revealed further decreases in skeletal oxygen concentrations upon S. aureus infection. The fitness of an srrAB mutant during osteomyelitis was significantly increased by depletion of neutrophils, suggesting that neutrophils impose hypoxic and/or nitrosative stresses on invading bacteria. To more globally evaluate staphylococcal responses to changing oxygenation, we examined quorum sensing and virulence factor production in staphylococci grown under aerobic or hypoxic conditions. Hypoxic growth resulted in a profound increase in quorum sensing-dependent toxin production, and a concomitant increase in cytotoxicity toward mammalian cells. Moreover, aerobic growth limited quorum sensing and cytotoxicity in an SrrAB-dependent manner, suggesting a mechanism by which S. aureus modulates quorum sensing and toxin production in response to environmental oxygenation. Collectively, our results demonstrate that bacterial hypoxic responses are key determinants of the staphylococcal-host interaction.

Activation of Hepatic STAT3 Maintains Pulmonary Defense during Endotoxemia.

Pneumonia and infection-induced sepsis are worldwide public health concerns. Both pathologies elicit systemic inflammation and induce a robust acute-phase response (APR). Although APR activation is well regarded as a hallmark of infection, the direct contributions of liver activation to pulmonary defense during sepsis remain unclear. By targeting STAT3-dependent acute-phase changes in the liver, we evaluated the role of liver STAT3 activity in promoting host defense in the context of sepsis and pneumonia. We employed a two-hit endotoxemia/pneumonia model, whereby administration of 18 h of intraperitoneal lipopolysaccharide (LPS; 5 mg/kg of body weight) was followed by intratracheal Escherichia coli (10(6) CFU) in wild-type mice or those lacking hepatocyte STAT3 (hepSTAT3(-/-)). Pneumonia alone (without endotoxemia) was effectively controlled in the absence of liver STAT3. Following endotoxemia and pneumonia, however, hepSTAT3(-/-) mice, with significantly reduced levels of circulating and airspace acute-phase proteins, exhibited significantly elevated lung and blood bacterial burdens and mortality. These data suggested that STAT3-dependent liver responses are necessary to promote host defense. While neither recruited airspace neutrophils nor lung injury was altered in endotoxemic hepSTAT3(-/-) mice, alveolar macrophage reactive oxygen species generation was significantly decreased. Additionally, bronchoalveolar lavage fluid from this group of hepSTAT3(-/-) mice allowed greater bacterial growth ex vivo. These results suggest that hepatic STAT3 activation promotes both cellular and humoral lung defenses. Taken together, induction of liver STAT3-dependent gene expression programs is essential to countering the deleterious consequences of sepsis on pneumonia susceptibility.

Induction of STAT3-Dependent CXCL5 Expression and Neutrophil Recruitment by Oncostatin-M during Pneumonia.

Acute bacterial pneumonia is a significant public health concern worldwide. Understanding the signals coordinating lung innate immunity may foster the development of therapeutics that limit tissue damage and promote host defense. We have previously shown that lung messenger RNA expression of the IL-6 family cytokine oncostatin-M (OSM) is significantly elevated in response to bacterial stimuli. However, its physiological significance during pneumonia is unknown. Here we demonstrate that OSM is rapidly increased in the airspaces of mice after pulmonary infection with Escherichia coli. Neutralization of OSM caused a substantial decrease in airspace neutrophils and macrophages. OSM blockade also caused a marked reduction in lung chemokine (C-X-C motif) ligand (CXCL) 5 expression, whereas other closely related neutrophil chemokines, CXCL1 and CXCL2, were unaffected. Intratracheal administration of recombinant OSM was sufficient to recapitulate the effect on CXCL5 induction, associated with robust activation of the signal transducer and activator of transcription 3 (STAT3) transcription factor. Cell sorting revealed that OSM effects were specific to lung epithelial cells, including a positive feedback loop in which OSM may facilitate expression of its own receptor. Finally, in vitro studies demonstrated that STAT3 was required for maximal OSM-induced CXCL5 expression. These studies demonstrate a novel role for OSM during pneumonia as an important signal to epithelial cells for chemokine induction mediating neutrophil recruitment.

The Lung-Liver Axis: A Requirement for Maximal Innate Immunity and Hepatoprotection during Pneumonia.

The hepatic acute-phase response (APR), stimulated by injury or inflammation, is characterized by significant changes in circulating acute-phase protein (APP) concentrations. Although individual functions of liver-derived APPs are known, the net consequence of APP changes is unclear. Pneumonia, which induces the APR, causes an inflammatory response within the airspaces that is coordinated largely by alveolar macrophages and is typified by cytokine production, leukocyte recruitment, and plasma extravasation, the latter of which may enable delivery of hepatocyte-derived APPs to the infection site. To determine the functional significance of the hepatic APR during pneumonia, we challenged APR-null mice lacking hepatocyte signal transducer and activator of transcription 3 (STAT3) and v-rel avian reticuloendotheliosis viral oncogene homolog A (RelA) with Escherichia coli in the airspaces. APR-null mice displayed ablated APP induction, significantly increased mortality, liver injury and apoptosis, and a trend toward increased bacterial burdens. TNF-α neutralization reversed hepatotoxicity, but not mortality, suggesting that APR-dependent survival is not solely due to hepatoprotection. After a milder (nonlethal) E. coli infection, hepatocyte-specific mutations decreased APP concentrations and pulmonary inflammation in bronchoalveolar lavage fluid. Cytokine expression in airspace macrophages, but not other airspace or circulating cells, was significantly dependent on APP extravasation into the alveoli. These data identify a novel signaling axis whereby the liver response enhances macrophage activation and pulmonary inflammation during pneumonia. Although hepatic acute-phase changes directly curb injury induced by TNF-α in the liver itself, APPs downstream of these same signals promote survival in association with innate immunity in the lungs, thus demonstrating a critical role for the lung-liver axis during pneumonia.

Cytotoxic Virulence Predicts Mortality in Nosocomial Pneumonia Due to Methicillin-Resistant Staphylococcus aureus.

The current study identified bacterial factors that may improve management of methicillin-resistant Staphylococcus aureus (MRSA) nosocomial pneumonia. Isolates were obtained from 386 patients enrolled in a randomized, controlled study of antibiotic efficacy. Isolates were screened for production of virulence factors and for vancomycin susceptibility. After adjustment for host factors such as severity of illness and treatment modality, cytotoxic activity was strongly and inversely associated with mortality; however, it had no effect on clinical cure. Isolates having low cytotoxicity, which were derived largely from healthcare-associated clones, exhibited a greater prevalence of vancomycin heteroresistance, and they were recovered more often from patients who were older and frailer. Additionally, a clone with low cytotoxic activity was associated with death and poor clinical improvement. Clone specificity and attenuated virulence appear to be associated with outcome. To our knowledge, these are the first correlations between MRSA virulence and mortality in nosocomial pneumonia.

Myeloid ZFP36L1 does not regulate inflammation or host defense in mouse models of acute bacterial infection.

Zinc finger protein 36, C3H type-like 1 (ZFP36L1) is one of several Zinc Finger Protein 36 (Zfp36) family members, which bind AU rich elements within 3' untranslated regions (UTRs) to negatively regulate the post-transcriptional expression of targeted mRNAs. The prototypical member of the family, Tristetraprolin (TTP or ZFP36), has been well-studied in the context of inflammation and plays an important role in repressing pro-inflammatory transcripts such as TNF-α. Much less is known about the other family members, and none have been studied in the context of infection. Using macrophage cell lines and primary alveolar macrophages we demonstrated that, like ZFP36, ZFP36L1 is prominently induced by infection. To test our hypothesis that macrophage production of ZFP36L1 is necessary for regulation of the inflammatory response of the lung during pneumonia, we generated mice with a myeloid-specific deficiency of ZFP36L1. Surprisingly, we found that myeloid deficiency of ZFP36L1 did not result in alteration of lung cytokine production after infection, altered clearance of bacteria, or increased inflammatory lung injury. Although alveolar macrophages are critical components of the innate defense against respiratory pathogens, we concluded that myeloid ZFP36L1 is not essential for appropriate responses to bacteria in the lungs. Based on studies conducted with myeloid-deficient ZFP36 mice, our data indicate that, of the Zfp36 family, ZFP36 is the predominant negative regulator of cytokine expression in macrophages. In conclusion, these results imply that myeloid ZFP36 may fully compensate for loss of ZFP36L1 or that Zfp36l1-dependent mRNA expression does not play an integral role in the host defense against bacterial pneumonia.

Negative elongation factor (NELF) coordinates RNA polymerase II pausing, premature termination, and chromatin remodeling to regulate HIV transcription.

A barrier to eradicating HIV infection is targeting and eliminating latently infected cells. Events that contribute to HIV transcriptional latency include repressive chromatin structure, transcriptional interference, the inability of Tat to recruit positive transcription factor b, and poor processivity of RNA polymerase II (RNAP II). In this study, we investigated mechanisms by which negative elongation factor (NELF) establishes and maintains HIV latency. Negative elongation factor (NELF) induces RNAP II promoter proximal pausing and limits provirus expression in HIV-infected primary CD4(+) T cells. Decreasing NELF expression overcomes RNAP II pausing to enhance HIV transcription elongation in infected primary T cells, demonstrating the importance of pausing in repressing HIV transcription. We also show that RNAP II pausing is coupled to premature transcription termination and chromatin remodeling. NELF interacts with Pcf11, a transcription termination factor, and diminishing Pcf11 in primary CD4(+) T cells induces HIV transcription elongation. In addition, we identify NCoR1-GPS2-HDAC3 as a NELF-interacting corepressor complex that is associated with repressed HIV long terminal repeats. We propose a model in which NELF recruits Pcf11 and NCoR1-GPS2-HDAC3 to paused RNAP II, reinforcing repression of HIV transcription and establishing a critical checkpoint for HIV transcription and latency.

Nasal carriage as a source of agr-defective Staphylococcus aureus bacteremia.

Inactivating mutations in the Staphylococcus aureus virulence regulator agr are associated with worse outcomes in bacteremic patients. However, whether agr dysfunction is primarily a cause or a consequence of early bacteremia is unknown. Analysis of 158 paired S. aureus clones from blood and nasal carriage sites in individual patients revealed that recovery of an agr-defective mutant from blood was usually predicted by the agr functionality of carriage isolates. Many agr-positive blood isolates produced low levels of hemolytic toxins, but levels were similar to those of colonizing strains within patients, suggesting that introduction into the blood did not select for mutations with minor functional effects. Evidently, the transition from commensalism to opportunism in S. aureus does not require full virulence in hospitalized patients. Furthermore, agr-defective mutants were found in uninfected nasal carriers in the same proportion as in carriers who develop bacteremia, suggesting low correlation between virulence and infectivity.

Real-time nucleic acid sequence-based amplification assay for rapid detection and quantification of agr functionality in clinical Staphylococcus aureus isolates.

Staphylococcus aureus infections are a significant cause of morbidity and mortality in health care settings. S. aureus clinical isolates vary in the function of the accessory gene regulator (agr), which governs the expression of virulence determinants, including surface and exoproteins, while agr activity has been correlated with patient outcome and treatment efficiency. Here we describe a duplex real-time nucleic acid sequence-based amplification (NASBA) detection and quantification platform for rapid determination of agr functionality in clinical isolates. Using the effector of agr response, RNAIII, as the assay target, and expression of the gyrase gene (gyrB) as a normalizer, we were able to accurately discriminate agr functionality in a single reaction. Time to positivity (TTP) ratios between gyrB and RNAIII showed very good correlation with the ratios of RNAIII versus gyrB RNA standard inputs and were therefore used as a simple readout to evaluate agr functionality. We validated the assay by characterizing 106 clinical S. aureus isolates, including strains with genetically characterized agr mutations. All isolates with dysfunctional agr activity exhibited a TTP ratio (TTP(gyrB)/TTP(RNAIII)) lower than 1.10, whereas agr-positive isolates had a TTP ratio higher than this value. The results showed that the assay was capable of determining target RNA ratios over 8 logs (10(-3) to 10(4)) with high sensitivity and specificity, suggesting the duplex NASBA assay may be useful for rapid determination of agr phenotypes and virulence potential in S. aureus clinical isolates.