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INTRODUCTION: Survivors of sepsis exhibit persistent immunosuppression. Epigenetic events may be responsible for some of these immunosuppressive changes. During sepsis circulating exosomes contain large quantities of DNA methyltransferase (DNMT) mRNAs. We hypothesized that exosomes directly transfer DNMT mRNAs to recipient monocytes with resultant methylation events and immunosuppression. METHODS: Exosomes containing DNMT mRNA were generated by stimulating monocytes with LPS. Confocal microscopy was used to determine uptake kinetics in the presence of pharmacologic inhibition. Expression and packaging of specific DNMT mRNA was controlled using DNMT siRNAs. Whole genome and gene specific methylation was assessed using bisulfite sequencing. Ingenuity pathway analysis was performed to determine the biological function of significance of differentially methylated regions. RESULTS: Exosomes effectively transferred DNMT mRNA to recipient monocytes. Pharmacologic inhibition of exosome uptake prevented this increase in DNMT mRNA expression. Recipient monocytes exhibited hypermethylation changes and gene suppression. siRNAs decreased the packaging of DNMT mRNAs and prevented TNFα gene suppression, restoring immunocompetence. CONCLUSION: These data support a role for exosome-mediated transfer of DNMT mRNA with resultant methylation and gene silencing. Pharmacologic uptake inhibition or targeted siRNA mediated DNMT gene silencing prevented DNMT mRNA transfer and maintained the cell’s ability to express TNFα in response to LPS. This highlights the potential therapeutic value of targeting these exosome-mediated epigenetic events to maintain the host immune response during sepsis.

ARDS is a common and highly lethal condition. The mortality has improved with supportive care over the past few decades, but the morbidity has not. Although there is no current treatment for ARDS that is based on molecular mechanisms, the science of ARDS is improving rapidly. The current pandemic has emphasized the urgent need for treatments that reduce the mortality and alleviate the morbidity of ARDS.

Mechanical ventilation generates injurious forces that exacerbate lung injury. These forces disrupt lung barrier integrity, trigger proinflammatory mediator release, and differentially regulate genes and non-coding oligonucleotides including microRNAs. In this study, we identify miR-146a as a mechanosensitive microRNA in alveolar macrophages that has therapeutic potential to mitigate lung injury during mechanical ventilation. We use humanized in-vitro systems, mouse models, and biospecimens from patients to elucidate the expression dynamics of miR-146a needed to decrease lung injury during mechanical ventilation. We find that the endogenous increase in miR-146a following injurious ventilation is not sufficient to prevent lung injury. However, when miR-146a is highly overexpressed using a nanoparticle delivery platform it is sufficient to prevent injury. These data indicate that the endogenous increase in microRNA-146a during mechanical ventilation is a compensatory response that partially limits injury and that nanoparticle delivery of miR-146a is an effective strategy for mitigating lung injury during mechanical ventilation., There are no targeted pharmacologic therapies to treat lung injury during mechanical ventilation (MV). Here the authors identify a mechanosensitive microRNA (miR-146a) in alveolar macrophages during MV and increase miR-146a to supraphysiological levels in these cells to mitigate ventilator induced lung injury.

Acute respiratory distress syndrome (ARDS) is an inflammatory lung disease with a high morbidity and mortality rate, for which no pharmacologic treatment is currently available. Our previous studies discovered that a pivotal step in the disease process is the activation of the nuclear factor of activated T cells (NFAT) c3 in lung macrophages, suggesting that inhibitors against the upstream protein phosphatase calcineurin should be effective for prevention/treatment of ARDS. Herein, we report the development of a highly potent, cell-permeable, and metabolically stable peptidyl inhibitor, CNI103, which selectively blocks the interaction between calcineurin and NFATc3, through computational and medicinal chemistry. CNI103 specifically inhibited calcineurin signaling in vitro and in vivo and exhibited a favorable pharmacokinetic profile, broad tissue distribution following different routes of administration, and minimal toxicity. Our data indicate that CNI103 is a promising novel treatment for ARDS and other inflammatory diseases.

Objectives This report provides analyses and perspective of a survey of critical care workforce, workload, and burnout among the intensivists and advanced practice providers of established U.S. and Canadian critical care organizations and provides a research agenda. Design A 97-item electronic survey questionnaire was distributed to the leaders of 27 qualifying organizations. Setting United States and Canada. Participants Leaders of critical care organizations in the United States and Canada. Interventions None. Data synthesis and main results We received 23 responses (85%). The critical care organization survey recorded substantial variability of most organizational aspects that were not restricted by the critical care organization definition or regulatory mandates. The most common physician staffing model was a combination of full-time and part-time intensivists. Approximately 80% of critical care organizations had dedicated advanced practice providers that staffed some or all their ICUs. Full-time intensivists worked a median of 168 days (range 42-192 d) in the ICU (168 shifts = 24 7-d wk). The median shift duration was 12 hours (range, 7-14 hr), and the median number of consecutive shifts allowed was 7 hours (range 7-14 hr). More than half of critical care organizations reported having burnout prevention programs targeted to ICU physicians, advanced practice providers, and nurses. Conclusions The variability of current approaches suggests that systematic comparative analyses could identify best organizational practices. The research agenda for the study of critical care organizations should include studies that provide insights regarding the effects of the integrative structure of critical care organizations on outcomes at the levels of our patients, our workforce, our work practices, and sustainability.

Renal tubular epithelial cells (RTECs) perform the essential function of maintaining the constancy of body fluid composition and volume. Toxic, inflammatory, or hypoxic-insults to RTECs can cause systemic fluid imbalance, electrolyte abnormalities and metabolic waste accumulation- manifesting as acute kidney injury (AKI), a common disorder associated with adverse long-term sequelae and high mortality. Here we report the results of a kinome-wide RNAi screen for cellular pathways involved in AKI-associated RTEC-dysfunction and cell death. Our screen and validation studies reveal an essential role of Cdkl5-kinase in RTEC cell death. In mouse models, genetic or pharmacological Cdkl5 inhibition mitigates nephrotoxic and ischemia-associated AKI. We propose that Cdkl5 is a stress-responsive kinase that promotes renal injury in part through phosphorylation-dependent suppression of pro-survival transcription regulator Sox9. These findings reveal a surprising non-neuronal function of Cdkl5, identify a pathogenic Cdkl5-Sox9 axis in epithelial cell-death, and support CDKL5 antagonism as a therapeutic approach for AKI.

Background: Patients with sepsis exhibit significant long-term immunosuppressive sequelae. Monocyte dysfunction is a hallmark of this damage. Circulating exosomes are an important mediator of the systemic signaling events that occur during the septic response; thus, we sought to characterize the contribution of circulating exosomes to the inflammatory process induced during sepsis Methods: Monocyte-derived exosomes were isolated from cultured monocytes from healthy adult donors via stimulation with lipopolysaccharide (LPS) or phosphate-buffered saline (PBS). The proteome was determined by capillary-liquid chromatography–nanospray tandem mass spectrometry (capillary–LC/NT/MS). Using pathway analysis, proteomic networks of exosomes derived from LPS-stimulated monocytes were compared with those isolated from patients with surgical sepsis. Naïve monocytes were then treated with these exosomes and stimulated with LPS to determine the effects on recipient-cell immune function. Results: Proteomic analysis demonstrated 18 differentially expressed proteins (17 down-regulated, one up-regulated) in sepsis-derived exosomes, with 15 differentially expressed proteins (14 down-regulated, one up-regulated) in the LPS-stimulated exosomes. Functional enrichment analysis demonstrated several down-regulated processes, including localization, biogenesis, and metabolic and cellular processes in addition to immune system processes. In LPS-stimulated macrophages, similar down-regulated processes were seen, including metabolic and cellular processes, as well as the response to stimulus. Cells treated with sepsis-derived exosomes or exosomes from LPS-stimulated monocytes demonstrated significant reductions in tumor necrosis factor (TNF)-α generation in response to LPS stimulation. Conclusions: Proteomic analysis of sepsis-derived exosomes and LPS-stimulated, macrophage-derived exosomes exhibited down-regulation of several important protein networks, including the immune response. In addition, human monocytes treated with exosomes from patients with sepsis or LPS-stimulated monocytes demonstrated significant reductions in TNF-α generation in response to LPS stimulation. These data suggest the contribution of circulating exosomes to systemic signaling and immunomodulation during sepsis.

Both hyperoxemia and hypoxemia are deleterious in critically ill patients. Targeted oxygenation is recommended to prevent both of these extremes, however this has not translated to the bedside. Hyperoxemia likely persists more than hypoxemia due to absence of immediate discernible adverse effects, cognitive biases and delay in prioritization of titration.We present the methodology for the Titration Of Oxygen Levels (TOOL) trial, an open label, randomized controlled trial of an algorithm-based FiOThis study is designed to evaluate the efficacy of a high fidelity, bioinformatics-based, electronic medical record derived electronic alert system to improve targeted oxygenation in mechanically ventilated patients by reducing excessive FiO

Background A new approach targeting aeroallergen sensing in the early events of mucosal immunity could have greater benefit. The CSF1-CSF1R pathway has a critical role in trafficking allergens to regional lymph nodes through activating dendritic cells. Intervention in this pathway could prevent allergen sensitization and subsequent Th2 allergic inflammation. Objective To examine the therapeutic effectiveness of CSF1 and CSF1R inhibition for blocking the dendritic cell function of sensing aeroallergens. Methods We adopted a model of chronic asthma induced by a panel of three naturally occurring allergens and novel delivery system of CSF1R inhibitor encapsulated nanoprobe. Results Selective depletion of CSF1 in airway epithelial cells abolished the production of allergen-reactive IgE, resulting in prevention of new asthma development as well as reversal of established allergic lung inflammation. CDPL-GW nanoprobe containing GW2580, a selective CSF1R inhibitor, showed favorable pharmacokinetics for inhalational treatment and intranasal insufflation delivery of CDPL-GW nanoprobe ameliorated asthma pathologies including allergen-specific serum IgE production, allergic lung and airway inflammation and airway hyper-responsiveness (AHR) with minimal pulmonary adverse reaction. Conclusion The inhibition of the CSF1-CSF1R signaling pathway effectively suppresses sensitization to aeroallergens and consequent allergic lung inflammation in a murine model of chronic asthma. CSF1R inhibition is a promising new target for the treatment of allergic asthma.

The incidence of asthma has increased from 5.5% to near 8% of the population, which is a major health concern. The hallmarks of asthma include eosinophilic airway inflammation that is associated with chronic airway remodeling. Allergic airway inflammation is characterized by a complex interplay of resident and inflammatory cells. MicroRNAs (miRNAs) are small noncoding RNAs that function as posttranscriptional modulators of gene expression. However, the role of miRNAs, specifically miR-451, in the regulation of allergic airway inflammation is unexplored. Our previous findings showed that oxidant stress regulates miR-451 gene expression in macrophages during an inflammatory process. In this paper, we examined the role of miR-451 in regulating macrophage phenotype using an experimental poly-allergenic murine model of allergic airway inflammation. We found that miR-451 contributes to the allergic induction of CCL17 in the lung and plays a key role in proasthmatic macrophage activation. Remarkably, administration of a Sirtuin 2 (Sirt2) inhibitor diminished alternate macrophage activation and markedly abrogated triple-allergen [dust mite, ragweed, Aspergillus fumigatus (DRA)]-induced lung inflammation. These data demonstrate a role for miR-451 in modulating allergic inflammation by influencing allergen-mediated macrophages phenotype.

Hypoxia leading to stabilization of hypoxia-inducible factor 1α (HIF-1α) serves as an early upstream initiator for adipose tissue (AT) dysfunction. Monocyte-derived macrophage infiltration in AT contributes to inflammation, fibrosis and obesity-related metabolic dysfunction. It was previously reported that myeloid cell-specific deletion of Hif-1α protected against high-fat diet (HFD)-induced AT dysfunction. Prolyl hydroxylases (PHDs) are key regulators of HIF-1α. We examined the effects of myeloid cell-specific upregulation and stabilization of Hif-1α via deletion of prolyl-hydroxylase 2 ( Phd2) and whether interleukin-1 receptor associated kinase-M ( Irak-M), a known downstream target of Hif-1α, contributes to Hif-1α-induced AT dysfunction. Our data show that with HFD, Hif-1α and Irak-M expressions were increased in the AT macrophages of Phd2flox/flox/ LysMcre mice compared with LysMcre mice. With HFD, Phd2flox/flox/ LysMcre mice exhibited increased AT inflammation, fibrosis, and systemic insulin resistance compared with control mice. Furthermore, Phd2flox/flox/ LysMcre mice bone marrow-derived macrophages exposed to hypoxia in vitro also had increased expressions of both Hif-1α and Irak-M. In wild-type mice, HFD induced upregulation of both HIF-1a and Irak-M in adipose tissue. Despite equivalent expression of Hif-1α compared with wild-type mice, globally-deficient Irak-M mice fed a HFD exhibited less macrophage infiltration, decreased inflammation and fibrosis and improved glucose tolerance. Global Irak-M deficiency was associated with an alternatively-activated macrophage phenotype in the AT after HFD. Together, these data show for the first time that an Irak-M-dependent mechanism likely mediates obesity-related AT dysfunction in conjunction with Hif-1α upregulation.

Acute respiratory distress syndrome (ARDS) is a highly lethal condition that impairs lung function and causes respiratory failure. Mechanical ventilation maintains gas exchange in patients with ARDS, but exposes lung cells to physical forces that exacerbate lung injury. Our data demonstrate that mTOR complex 1 (mTORC1) is a mechanosensor in lung epithelial cells and that activation of this pathway during mechanical ventilation exacerbates lung injury. We found that mTORC1 is activated in lung epithelial cells following volutrauma and atelectrauma in mice and humanized in vitro models of the lung microenvironment. mTORC1 is also activated in lung tissue of mechanically ventilated patients with ARDS. Deletion of Tsc2, a negative regulator of mTORC1, in epithelial cells exacerbates physiologic lung dysfunction during mechanical ventilation. Conversely, treatment with rapamycin at the time mechanical ventilation is initiated prevents physiologic lung injury (i.e. decreased compliance) without altering lung inflammation or barrier permeability. mTORC1 inhibition mitigates physiologic lung injury by preventing surfactant dysfunction during mechanical ventilation. Our data demonstrate that in contrast to canonical mTORC1 activation under favorable growth conditions, activation of mTORC1 during mechanical ventilation exacerbates lung injury and inhibition of this pathway may be a novel therapeutic target to mitigate ventilator induced lung injury during ARDS.

Background The pathogenesis of asthma and airway obstruction is the result of an abnormal response to different environmental exposures. The scientific premise of our study was based on the finding that FoxO1 expression is increased in lung macrophages of mice after allergen exposure and human asthmatic patients. Macrophages are capable of switching from one functional phenotype to another, and it is important to understand the mechanisms involved in the transformation of macrophages and how their cellular function affects the peribronchial stromal microenvironment. Methods We employed a murine asthma model, in which mice were treated by intranasal insufflation with allergens for 2-8 weeks. We used both a pharmacologic approach using a highly specific FoxO1 inhibitor and genetic approaches using FoxO1 knockout mice (FoxO1fl/fl LysMcre). Cytokine level in biological fluids was measured by ELISA and the expression of encoding molecules by NanoString assay and qRT-PCR. Results We show that the levels of FoxO1 gene are significantly elevated in the airway macrophages of patients with mild asthma in response to subsegmental bronchial allergen challenge. Transcription factor FoxO1 regulates a pro-asthmatic phenotype of lung macrophages that is involved in the development and progression of chronic allergic airway disease. We have shown that inhibition of FoxO1 induced phenotypic conversion of lung macrophages and downregulates pro-asthmatic and pro-fibrotic gene expression by macrophages, which contribute to airway inflammation and airway remodeling in allergic asthma. Conclusion Targeting FoxO1 with its downstream regulator IRF4 is a novel therapeutic target for controlling allergic inflammation and potentially reversing fibrotic airway remodeling.

International audience; AbstractAmong invertebrates, cellular innate immunity is critical for wound healing and defense against parasites and pathogens. While the study of cellular immunity has received much attention in model insects, the study of hemocytes, including immune cells, in honey bees has received little attention. Much of our understanding of honey bee hemocytes is derived from a limited set of methodologies, predominately utilizing bright-field microscopy, which makes broad conclusions about honey bee cellular immunity difficult to infer. We build upon existing methodologies using differential cell staining, in vitro phagocytosis assays, and an analysis of respiratory burst activity as measured through reactive oxygen species (ROS) production. Further, we characterize the morphological diversity and functional capacity of honey bee hemocytes in both adult workers and young queen bees as well as the ontogeny of the hemocyte population across larval and adult stages of the worker caste. Our findings suggest that granulocytes are the major phagocytic cells in honey bees and that circulating larval granulocytes undergo mitotic cell division. Additionally, we demonstrate that ROS production in larval hemocytes can be stimulated with the protein kinase C activator, phorbol 12-myristate 13-acetate. This indicates the presence of a functional protein kinase C-dependent phagocyte oxidase system, though further experimentation is needed to confirm phagocyte oxidase as the source of ROS. Overall, this work expands our knowledge of honey bee hemocytes and provides additional methodological tools for studying immune mechanisms in insects.

Specific therapies targeting cellular and molecular events of sepsis induced Acute Lung Injury (ALI) pathogenesis are lacking. We have reported a pivotal role for Nuclear Factors of Activated T cells (NFATc3) in regulating macrophage phenotype during sepsis induced ALI and subsequent studies demonstrate that NFATc3 transcriptionally regulates macrophage CCR2 and TNFα gene expression. Mouse pulmonary microvascular endothelial cell monolayer maintained a tighter barrier function when co-cultured with LPS stimulated NFATc3 deficient macrophages whereas wild type macrophages caused leaky monolayer barrier. More importantly, NFATc3 deficient mice showed decreased neutrophilic lung inflammation, improved alveolar capillary barrier function, arterial oxygen saturation and survival benefit in lethal CLP sepsis mouse models. In addition, survival of wild type mice subjected to the lethal CLP sepsis was not improved with broad-spectrum antibiotics, whereas the survival of NFATc3 deficient mice was improved to 40-60% when treated with imipenem. Passive adoptive transfer of NFATc3 deficient macrophages conferred protection against LPS induced ALI in wild type mice. Furthermore, CP9-ZIZIT, a highly potent, cell-permeable peptide inhibitor of Calcineurin inhibited NFATc3 activation. CP9-ZIZIT effectively reduced sepsis induced inflammatory cytokines and pulmonary edema in mice. Thus, this study demonstrates that inhibition of NFATc3 activation by CP9-ZIZIT provides a potential therapeutic option for attenuating sepsis induced ALI/pulmonary edema.

During mechanical ventilation, injurious biophysical forces exacerbate lung injury. These forces disrupt alveolar capillary barrier integrity, trigger proinflammatory mediator release, and differentially regulate genes and non-coding oligonucleotides such as microRNAs. In this study, we identify miR-146a as a mechanosensitive microRNA in alveolar macrophages that has therapeutic potential to mitigate lung injury during mechanical ventilation. We used humanized in-vitro systems, mouse models, and biospecimens from mechanically ventilated patients to elucidate the expression dynamics of miR-146a that might be required to decrease lung injury during mechanical ventilation. We found that the endogenous increase in miR-146a following injurious was relatively modest and not sufficient to prevent lung injury. However, when miR-146a was highly overexpressed using a nanoparticle-based delivery platform in vivo, it was sufficient to prevent lung injury. These data indicate that the endogenous increase in microRNA-146a during MV is a compensatory response that only partially limits VILI and that nanoparticle delivery approaches that significantly over-express microRNA-146a in AMs is an effective strategy for mitigating VILI.

Synovial macrophages are crucial in the development of joint inflammation and bone damage; however, the pathways that control macrophage remodeling in inflammatory M1 cells or bone-eroding osteoclasts are not fully understood. We determined that elevated IL-7R/CD127 expression is the hallmark of rheumatoid arthritis (RA) M1 macrophages and that these cells are highly responsive to interleukin-7 (IL-7)-driven osteoclastogenesis. We established that lipopolysaccharide (LPS), interferon-γ (IFNγ), and tumor necrosis factor-α (TNFα), the classic M1 macrophage mediators, enhance IL-7R expression in RA and murine macrophages. The local expression of IL-7 provokes arthritis, predominantly through escalating the number of F480(+)iNOS(+) cells rather than CD3(+) T cells. Ectopic LPS injection stabilizes IL-7-induced arthritis by increasing myeloid IL-7R expression, in part via IFNγ induction. Hence, in RAG−/− mice, IL-7-mediated arthritis is suppressed because of the reduction in myeloid IL-7R expression due to the lack of IFNγ. Moreover, the amelioration of IL-7-induced arthritis by anti-TNF therapy is due to a decrease in the number of cells in the unique F480(+)iNOS(+)IL-7R(+)CCL5(+) subset, with no impact on the F480(+)Arginase(+) cell or CD3(+) T cell frequency. Consistent with the preclinical findings, the findings of a phase 4 study performed with RA patients following 6 months of anti-TNF therapy revealed that IL-7R expression was reduced without affecting the levels of IL-7. This study shifts the paradigm by discovering that IL-7-induced arthritis is dependent on F480(+)iNOS(+)IL-7R(+)CCL5(+) cell function, which activates TH-1 cells to amplify myeloid IL-7R expression and disease severity.

Idiopathic pulmonary fibrosis is a deadly disease characterized by excessive extracellular matrix deposition in the lungs, resulting in decreased pulmonary function. Although epithelial cells and fibroblasts have long been the focus of idiopathic pulmonary fibrosis research, the role of various subpopulations of macrophages in promoting a fibrotic response is an emerging target. Healthy lungs are composed of two macrophage populations, tissue-resident alveolar macrophages and interstitial macrophages, which help to maintain homeostasis. After injury, tissue-resident alveolar macrophages are depleted, and monocytes from the bone marrow (BM) traffic to the lungs along a CCL2/CCR2 axis and differentiate into monocyte-derived alveolar macrophages (Mo-AMs), which is a cell population implicated in murine models of pulmonary fibrosis. In this study, we sought to determine how IL-1R–associated kinase-M (IRAK-M), a negative regulator of TLR signaling, modulates monocyte trafficking into the lungs in response to bleomycin. Our data indicate that after bleomycin challenge, mice lacking IRAK-M have decreased monocyte trafficking and reduced Mo-AMs in their lungs. Although IRAK-M expression did not regulate differences in chemokines, cytokines, or adhesion molecules associated with monocyte recruitment, IRAK-M was necessary for CCR2 upregulation following bleomycin challenge. This finding prompted us to develop a competitive BM chimera model, which demonstrated that expression of BM-derived IRAK-M was necessary for monocyte trafficking into the lung and for subsequent enhanced collagen deposition. These data indicate that IRAK-M regulates monocyte trafficking by increasing the expression of CCR2, resulting in enhanced monocyte translocation into the lung, Mo-AM differentiation, and development of pulmonary fibrosis.

BackgroundTreatment with bone-marrow-derived mesenchymal stromal cells (MSCs) has shown benefits in preclinical models of acute respiratory distress syndrome (ARDS). Safety has not been established for administration of MSCs in critically ill patients with ARDS. We did a phase 2a trial to assess safety after administration of MSCs to patients with moderate to severe ARDS.MethodsWe did a prospective, double-blind, multicentre, randomised trial to assess treatment with one intravenous dose of MSCs compared with placebo. We recruited ventilated patients with moderate to severe ARDS (ratio of partial pressure of oxygen to fractional inspired oxygen

To assess-by literature review and expert consensus-workforce, workload, and burnout considerations among intensivists and advanced practice providers.Data were synthesized from monthly expert consensus and literature review.Workforce and Workload section workgroup of the Academic Leaders in Critical Care Medicine Task Force.Multidisciplinary care teams led by intensivists are an essential component of critical care delivery. Advanced practice providers (nurse practitioners and physician assistants) are progressively being integrated into ICU practice models. The ever-increasing number of patients with complex, life-threatening diseases, concentration of ICU beds in few centralized hospitals, expansion of specialty ICU services, and desire for 24/7 availability have contributed to growing intensivist staffing concerns. Such staffing challenges may negatively impact practitioner wellness, team perception of care quality, time available for teaching, and length of stay when the patient to intensivist ratio is greater than or equal to 15. Enhanced team communication and reduction of practice variation are important factors for improved patient outcomes. A diverse workforce adds value and enrichment to the overall work environment. Formal succession planning for ICU leaders is crucial to the success of critical care organizations. Implementation of a continuous 24/7 ICU coverage care model in high-acuity, high-volume centers should be based on patient-centered outcomes. High levels of burnout syndrome are common among intensivists. Prospective analyses of interventions to decrease burnout within the ICU setting are limited. However, organizational interventions are felt to be more effective than those directed at individuals.Critical care workforce and staffing models are myriad and based on several factors including local culture and resources, ICU organization, and strategies to reduce burden on the ICU provider workforce. Prospective studies to assess and avoid the burnout syndrome among intensivists and advanced practice providers are needed.

We have reported that von Hippel-Lindau protein (pVHL) expression is elevated in human and mouse fibrotic lungs and that overexpression of pVHL stimulates fibroblast proliferation. We sought to determine whether loss of pVHL in fibroblasts prevents injury and fibrosis in mice that are treated with bleomycin. We generated heterozygous fibroblast-specific pVHL (Fsp-VHL) knockdown mice (Fsp-VHL(+/-)) and homozygous Fsp-VHL knockout mice (Fsp-VHL(-/-)) by crossbreeding vhlh 2-lox mice (VHL(fl/fl)) with Fsp-Cre recombinase mice. Our data show that Fsp-VHL(-/-) mice, but not Fsp-VHL(+/-) mice, have elevated red blood cell counts, hematocrit, hemoglobin content, and expression of hypoxia-inducible factor (HIF) targets, indicating HIF activation. To examine the role of pVHL in bleomycin-induced lung injury and fibrosis in vivo, we administered PBS or bleomycin to age-, sex-, and strain-matched 8-week-old VHL(fl/fl), Fsp-VHL(+/-), and Fsp-VHL(-/-) mice. In Fsp-VHL(+/-) and Fsp-VHL(-/-) mice, bleomycin-induced collagen accumulation, fibroblast proliferation, differentiation, and matrix protein dysregulation were markedly attenuated. Suppression of pVHL also decreased bleomycin-induced Wnt signaling and prostaglandin E2 signaling but did not affect bleomycin-induced initial acute lung injury and lung inflammation. These results indicate that pVHL has a pivotal role in bleomycin-induced pulmonary fibrosis, possibly via an HIF-independent pathway. Paradoxically, pVHL does not affect bleomycin-induced lung injury and inflammation, indicating a separation of the mechanisms involved in injury/inflammation from those involved in pulmonary fibrosis.

Inflammatory monocyte and tissue macrophages influence the initiation, progression, and resolution of type 2 immune responses, and alveolar macrophages are the most prevalent immune-effector cells in the lung. While we were characterizing the M1- or M2-like macrophages in type 2 allergic inflammation, we discovered that FoxO1 is highly expressed in alternatively activated macrophages. Although several studies have been focused on the fundamental role of FoxOs in hematopoietic and immune cells, the exact role that FoxO1 plays in allergic asthmatic inflammation in activated macrophages has not been investigated. Growing evidences indicate that FoxO1 acts as an upstream regulator of IRF4 and could have a role in a specific inflammatory phenotype of macrophages. Therefore, we hypothesized that IRF4 expression regulated by FoxO1 in alveolar macrophages is required for established type 2 immune mediates allergic lung inflammation. Our data indicate that targeted deletion of FoxO1 using FoxO1-selective inhibitor AS1842856 and genetic ablation of FoxO1 in macrophages significantly decreases IRF4 and various M2 macrophage-associated genes, suggesting a mechanism that involves FoxO1-IRF4 signaling in alveolar macrophages that works to polarize macrophages toward established type 2 immune responses. In response to the challenge of DRA (dust mite, ragweed, and Aspergillus) allergens, macrophage specific FoxO1 overexpression is associated with an accentuation of asthmatic lung inflammation, whereas pharmacologic inhibition of FoxO1 by AS1842856 attenuates the development of asthmatic lung inflammation. Thus, our study identifies a role for FoxO1-IRF4 signaling in the development of alternatively activated alveolar macrophages that contribute to type 2 allergic airway inflammation.

Alveolar epithelial and endothelial cell injury is a major feature of the acute respiratory distress syndrome, in particular when in conjunction with ventilation therapies. Previously we showed [Kim SC, Kellett T, Wang S, Nishi M, Nagre N, Zhou B, Flodby P, Shilo K, Ghadiali SN, Takeshima H, Hubmayr RD, Zhao X. Am J Physiol Lung Cell Mol Physiol 307: L449–L459, 2014.] that tripartite motif protein 72 (TRIM72) is essential for amending alveolar epithelial cell injury. Here, we posit that TRIM72 improves cellular integrity through its interaction with caveolin 1 (Cav1). Our data show that, in primary type I alveolar epithelial cells, lack of TRIM72 led to significant reduction of Cav1 at the plasma membrane, accompanied by marked attenuation of caveolar endocytosis. Meanwhile, lentivirus-mediated overexpression of TRIM72 selectively increases caveolar endocytosis in rat lung epithelial cells, suggesting a functional association between these two. Further coimmunoprecipitation assays show that deletion of either functional domain of TRIM72, i.e., RING, B-box, coiled-coil, or PRY-SPRY, abolishes the physical interaction between TRIM72 and Cav1, suggesting that all theoretical domains of TRIM72 are required to forge a strong interaction between these two molecules. Moreover, in vivo studies showed that injurious ventilation-induced lung cell death was significantly increased in knockout (KO) TRIM72KO and Cav1KO lungs compared with wild-type controls and was particularly pronounced in double KO mutants. Apoptosis was accompanied by accentuation of gross lung injury manifestations in the TRIM72KO and Cav1KO mice. Our data show that TRIM72 directly and indirectly modulates caveolar endocytosis, an essential process involved in repair of lung epithelial cells through removal of plasma membrane wounds. Given TRIM72's role in endomembrane trafficking and cell repair, we consider this molecule an attractive therapeutic target for patients with injured lungs.

Aberrant cell cycle activation is a hallmark of carcinogenesis. Recently three cell cycle targeting cyclin-dependent kinase 4/6 (CDK4/6) inhibitors have been approved for the treatment of metastatic breast cancer. CDK4/6 inhibitors suppress proliferation through inhibition of CDK4/6-dependent retinoblastoma-1 (Rb1) phosphorylation and inactivation, a key regulatory step in G1-to-S-phase transition. Importantly, aberrant cell cycle activation is also linked with several non-oncological diseases including acute kidney injury (AKI). AKI is a common disorder caused by toxic, inflammatory, and ischemic damage to renal tubular epithelial cells (RTECs). Interestingly, AKI triggered by the anti-cancer drug cisplatin can be mitigated by ribociclib, a CDK4/6 inhibitor, through mechanisms that remain unclear. Employing in vivo cell cycle analysis and functional Rb1 knock-down, here, we have examined the cellular and pharmacological basis of the renal protective effects of ribociclib during cisplatin nephrotoxicity. Remarkably, siRNA-mediated Rb1 silencing or RTEC-specific Rb1 gene ablation did not alter the severity of cisplatin-associated AKI; however, it completely abrogated the protective effects conferred by ribociclib administration. Furthermore, we find that cisplatin treatment evokes CDK4/6 activation and Rb1 phosphorylation in the normally quiescent RTECs, however, this is not followed by S-phase entry likely due to DNA-damage induced G1 arrest. The cytoprotective effects of ribociclib are thus not a result of suppression of S-phase entry but are likely dependent on the maintenance of Rb1 in a hypo-phosphorylated and functionally active form under stress conditions. These findings delineate the role of Rb1 in AKI and illustrate the pharmacological basis of the renal protective effects of CDK4/6 inhibitors.

Ischemic tissue damage activates hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM)-generating myeloid cells, and persistent HSPC activity may drive chronic inflammation and impair tissue recovery. Although increased reactive oxygen species in the BM regulate HSPC functions, their roles in myelopoiesis of activated HSPCs and subsequent tissue recovery during ischemic damage are not well understood. In this paper, we report that deletion of Nox2 NADPH oxidase in mice results in persistent elevations in BM HSPC activity and levels of inflammatory monocytes/macrophages in BM and ischemic tissue in a model of hindlimb ischemia. Ischemic tissue damage induces oxidants in BM such as elevations of hydrogen peroxide and oxidized phospholipids, which activate redox-sensitive Lyn kinase in a Nox2-dependent manner. Moreover, during tissue recovery after ischemic injury, this Nox2-ROS–Lyn kinase axis is induced by Nox2 in neutrophils that home to the BM, which inhibits HSPC activity and inflammatory monocyte generation and promotes tissue regeneration after ischemic damage. Thus, oxidant signaling in the BM mediated by Nox2 in neutrophils regulates myelopoiesis of HSPCs to promote regeneration of damaged tissue.

Allergic eosinophilic asthma is a chronic condition causing airway remodeling resulting in lung dysfunction. We observed that expression of sirtuin 2 (Sirt2), a histone deacetylase, regulates the recruitment of eosinophils after sensitization and challenge with a triple antigen: dust mite, ragweed, and Aspergillus fumigatus (DRA). Our data demonstrate that IL-4 regulates the expression of Sirt2 isoform 3/5. Pharmacological inhibition of Sirt2 by AGK2 resulted in diminished cellular recruitment, decreased CCL17/TARC, and reduced goblet cell hyperplasia. YM1 and Fizz1 expression was reduced in AGK2-treated, IL-4-stimulated lung macrophages in vitro as well as in lung macrophages from AGK2-DRA-challenged mice. Conversely, overexpression of Sirt2 resulted in increased cellular recruitment, CCL17 production, and goblet cell hyperplasia following DRA challenge. Sirt2 isoform 3/5 was upregulated in primary human alveolar macrophages following IL-4 and AGK2 treatment, which resulted in reduced CCL17 and markers of alternative activation. These gain-of-function and loss-of-function studies indicate that Sirt2 could be developed as a treatment for eosinophilic asthma.

High mobility group (HMG) proteins are a family of architectural transcription factors, with HMGA1 playing a role in the regulation of genes involved in promoting systemic inflammatory responses. We speculated that blocking HMGA1-mediated pathways might improve outcomes from sepsis. To investigate HMGA1 further, we developed genetically modified mice expressing a dominant negative (dn) form of HMGA1 targeted to the vasculature. In dnHMGA1 transgenic (Tg) mice, endogenous HMGA1 is present, but its function is decreased due to the mutant transgene. These mice allowed us to specifically study the importance of HMGA1 not only during a purely pro-inflammatory insult of endotoxemia, but also during microbial sepsis induced by implantation of a bacterial-laden fibrin clot into the peritoneum. We found that the dnHMGA1 transgene was only present in Tg and not wild-type (WT) littermate mice, and the mutant transgene was able to interact with transcription factors (such as NF-κB), but was not able to bind DNA. Tg mice exhibited a blunted hypotensive response to endotoxemia, and less mortality in microbial sepsis. Moreover, Tg mice had a reduced inflammatory response during sepsis, with decreased macrophage and neutrophil infiltration into tissues, which was associated with reduced expression of monocyte chemotactic protein-1 and macrophage inflammatory protein-2. Collectively, these data suggest that targeted expression of a dnHMGA1 transgene is able to improve outcomes in models of endotoxin exposure and microbial sepsis, in part by modulating the immune response and suggest a novel modifiable pathway to target therapeutics in sepsis.