Single-cell RNA sequencing uncovers the nuclear decoy lincRNA PIRAT as a regulator of systemic monocyte immunity during COVID-19.

The systemic immune response to viral infection is shaped by master transcription factors, such as NF-κB, STAT1, or PU.1. Although long noncoding RNAs (lncRNAs) have been suggested as important regulators of transcription factor activity, their contributions to the systemic immunopathologies observed during SARS-CoV-2 infection have remained unknown. Here, we employed a targeted single-cell RNA sequencing approach to reveal lncRNAs differentially expressed in blood leukocytes during severe COVID-19. Our results uncover the lncRNA PIRAT (PU.1-induced regulator of alarmin transcription) as a major PU.1 feedback-regulator in monocytes, governing the production of the alarmins S100A8/A9, key drivers of COVID-19 pathogenesis. Knockout and transgene expression, combined with chromatin-occupancy profiling, characterized PIRAT as a nuclear decoy RNA, keeping PU.1 from binding to alarmin promoters and promoting its binding to pseudogenes in naïve monocytes. NF-κB-dependent PIRAT down-regulation during COVID-19 consequently releases a transcriptional brake, fueling alarmin production. Alarmin expression is additionally enhanced by the up-regulation of the lncRNA LUCAT1, which promotes NF-κB-dependent gene expression at the expense of targets of the JAK-STAT pathway. Our results suggest a major role of nuclear noncoding RNA networks in systemic antiviral responses to SARS-CoV-2 in humans.

Lactate induces metabolic and epigenetic reprogramming of pro-inflammatory Th17 cells.

Increased lactate levels in the tissue microenvironment are a well-known feature of chronic inflammation. However, the role of lactate in regulating T cell function remains controversial. Here, we demonstrate that extracellular lactate predominantly induces deregulation of the Th17-specific gene expression program by modulating the metabolic and epigenetic status of Th17 cells. Following lactate treatment, Th17 cells significantly reduced their IL-17A production and upregulated Foxp3 expression through ROS-driven IL-2 secretion. Moreover, we observed increased levels of genome-wide histone H3K18 lactylation, a recently described marker for active chromatin in macrophages, in lactate-treated Th17 cells. In addition, we show that high lactate concentrations suppress Th17 pathogenicity during intestinal inflammation in mice. These results indicate that lactate is capable of reprogramming pro-inflammatory T cell phenotypes into regulatory T cells.

Streptococcus pneumoniae disrupts the structure of the golgi apparatus and subsequent epithelial cytokine response in an H2O2-dependent manner.

BACKGROUND: Lung infections caused by Streptococcus pneumonia are a global leading cause of death. The reactive oxygen species H2O2 is one of the virulence factors of Streptococcus pneumoniae. The Golgi apparatus is essential for the inflammatory response of a eukaryotic cell. Golgi fragmentation was previously shown to be induced by bacterial pathogens and in response to H2O2 treatment. This led us to investigate whether the Golgi apparatus is actively involved and targeted in host-pathogen interactions during pneumococcal infections. METHODS: Following in vitro infection of BEAS-2B bronchial epithelial cells with Streptococcus pneumoniae for 16 h, the structure of the Golgi apparatus was assessed by fluorescence staining of the Golgi-associated protein, Golgin-97. To investigate the effect of H2O2 production on Golgi structure, BEAS-2B cells were treated with H2O2 or the H2O2 degrading enzyme Catalase, prior to Golgi staining. Artificial disruption of the Golgi apparatus was induced by treatment of cells with the GBF1 inhibitor, Golgicide A. A proinflammatory cellular response was induced by treatment of cells with the bacterial cell wall component and TLR4 ligand lipoteichoic acid. RESULTS: In vitro infection of bronchial epithelial cells with wild type Streptococcus pneumoniae led to a disruption of normal Golgi structure. Golgi fragmentation was not observed after deletion of the pneumococcal H2O2-producing gene, spxB, or neutralization of H2O2 by catalase treatment, but could be induced by H2O2 treatment. Streptococcus pneumoniae infection significantly reduced host cell protein glycosylation and artificial disruption of Golgi structure significantly reduced bacterial adherence, but increased bacterial counts in the supernatant. To understand if this effect depended on cell-contact or soluble factors, pneumococci were treated with cell-supernatant of cells treated with Golgicide A and/or lipoteichoic acid. This approach revealed that lipoteichoic acid conditioned medium inhibits bacterial replication in presence of host cells. In contrast, artificial Golgi fragmentation by Golgicide A treatment prior to lipoteichoic acid treatment rescued bacterial replication. This effect was associated with an increase of IL-6 and IL-8 in the supernatant of lipoteichoic acid treated cells. The increased cytokine release was abolished if cells were treated with Golgicide A prior to lipoteichoic acid treatment. CONCLUSION: Streptococcus pneumoniae disrupts the Golgi apparatus in an H2O2-dependent manner, thereby inhibiting paracrine anti-infective mechanisms. Video Abstract.

Bacterial vesicles block viral replication in macrophages via TLR4-TRIF-axis.

Gram-negative bacteria naturally secrete nano-sized outer membrane vesicles (OMVs), which are important mediators of communication and pathogenesis. OMV uptake by host cells activates TLR signalling via transported PAMPs. As important resident immune cells, alveolar macrophages are located at the air-tissue interface where they comprise the first line of defence against inhaled microorganisms and particles. To date, little is known about the interplay between alveolar macrophages and OMVs from pathogenic bacteria. The immune response to OMVs and underlying mechanisms are still elusive. Here, we investigated the response of primary human macrophages to bacterial vesicles (Legionella pneumophila, Klebsiella pneumoniae, Escherichia coli, Salmonella enterica, Streptococcus pneumoniae) and observed comparable NF-κB activation across all tested vesicles. In contrast, we describe differential type I IFN signalling with prolonged STAT1 phosphorylation and strong Mx1 induction, blocking influenza A virus replication only for Klebsiella, E.coli and Salmonella OMVs. OMV-induced antiviral effects were less pronounced for endotoxin-free Clear coli OMVs and Polymyxin-treated OMVs. LPS stimulation could not mimic this antiviral status, while TRIF knockout abrogated it. Importantly, supernatant from OMV-treated macrophages induced an antiviral response in alveolar epithelial cells (AEC), suggesting OMV-induced intercellular communication. Finally, results were validated in an ex vivo infection model with primary human lung tissue. In conclusion, Klebsiella, E.coli and Salmonella OMVs induce antiviral immunity in macrophages via TLR4-TRIF-signaling to reduce viral replication in macrophages, AECs and lung tissue. These gram-negative bacteria induce antiviral immunity in the lung through OMVs, with a potential decisive and tremendous impact on bacterial and viral coinfection outcome. Video Abstract.

Surface proteome of plasma extracellular vesicles as mechanistic and clinical biomarkers for malaria.

PURPOSE: Malaria is a life-threatening mosquito-borne disease caused by Plasmodium parasites, mainly in tropical and subtropical countries. Plasmodium falciparum (P. falciparum) is the most prevalent cause on the African continent and responsible for most malaria-related deaths globally. Important medical needs are biomarkers for disease severity or disease outcome. A potential source of easily accessible biomarkers are blood-borne small extracellular vesicles (sEVs). METHODS: We performed an EV Array to find proteins on plasma sEVs that are differentially expressed in malaria patients. Plasma samples from 21 healthy subjects and 15 malaria patients were analyzed. The EV array contained 40 antibodies to capture sEVs, which were then visualized with a cocktail of biotin-conjugated CD9, CD63, and CD81 antibodies. RESULTS: We detected significant differences in the protein decoration of sEVs between healthy subjects and malaria patients. We found CD106 to be the best discrimination marker based on receiver operating characteristic (ROC) analysis with an area under the curve of > 0.974. Additional ensemble feature selection revealed CD106, Osteopontin, CD81, major histocompatibility complex class II DR (HLA-DR), and heparin binding EGF like growth factor (HBEGF) together with thrombocytes to be a feature panel for discrimination between healthy and malaria. TNF-R-II correlated with HLA-A/B/C as well as CD9 with CD81, whereas Osteopontin negatively correlated with CD81 and CD9. Pathway analysis linked the herein identified proteins to IFN-γ signaling. CONCLUSION: sEV-associated proteins can discriminate between healthy individuals and malaria patients and are candidates for future predictive biomarkers. TRIAL REGISTRATION: The trial was registered in the Deutsches Register Klinischer Studien (DRKS-ID: DRKS00012518).

Surface Proteome of Plasma Extracellular Vesicles as Biomarkers for Pneumonia and Acute Exacerbation of Chronic Obstructive Pulmonary Disease.

BACKGROUND: Community-acquired pneumonia (CAP) and acute exacerbation of chronic obstructive pulmonary disease (AECOPD) represent a major burden of disease and death and their differential diagnosis is critical. A potential source of relevant accessible biomarkers are blood-borne small extracellular vesicles (sEVs). METHODS: We performed an extracellular vesicle array to find proteins on plasma sEVs that are differentially expressed and possibly allow the differential diagnosis between CAP and AECOPD. Plasma samples were analyzed from 21 healthy controls, 24 patients with CAP, and 10 with AECOPD . The array contained 40 antibodies to capture sEVs, which were then visualized with a cocktail of biotin-conjugated CD9, CD63, and CD81 antibodies. RESULTS: We detected significant differences in the protein decoration of sEVs between healthy controls and patients with CAP or AECOPD. We found CD45 and CD28 to be the best discrimination markers between CAP and AECOPD in receiver operating characteristic analyses, with an area under the curve >0.92. Additional ensemble feature selection revealed the possibility to distinguish between CAP and AECOPD even if the patient with CAP had COPD, with a panel of CD45, CD28, CTLA4 (cytotoxic T-lymphocyte-associated protein 4), tumor necrosis factor-R-II, and CD16. CONCLUSION: The discrimination of sEV-associated proteins is a minimally invasive method with potential to discriminate between CAP and AECOPD.

Proviral MicroRNAs Detected in Extracellular Vesicles From Bronchoalveolar Lavage Fluid of Patients With Influenza Virus-Induced Acute Respiratory Distress Syndrome.

Influenza A virus (IAV) causes severe respiratory infections and alveolar epithelial damage resulting in acute respiratory distress syndrome (ARDS). Extracellular vesicles (EVs) have been shown to mediate cellular crosstalk in inflammation by transfer of microRNAs (miRNAs). In this study, we found significant changes in the miRNA composition of EVs in the bronchoalveolar lavage fluid from patients with IAV-induced ARDS. Among the 9 significantly deregulated microRNAs, miR-17-5p was upregulated in patients' BALF and in EVs of IAV-infected lung epithelial cells (A549). In these cells, transfer of miR-17-5p strongly downregulated expression of the antiviral factor Mx1 and significantly enhanced IAV replication.

An Interactive Macrophage Signal Transduction Map Facilitates Comparative Analyses of High-Throughput Data.

Macrophages (Mϕs) are key players in the coordination of the lifesaving or detrimental immune response against infections. The mechanistic understanding of the functional modulation of Mϕs by pathogens and pharmaceutical interventions at the signal transduction level is still far from complete. The complexity of pathways and their cross-talk benefits from holistic computational approaches. In the present study, we reconstructed a comprehensive, validated, and annotated map of signal transduction pathways in inflammatory Mϕs based on the current literature. In a second step, we selectively expanded this curated map with database knowledge. We provide both versions to the scientific community via a Web platform that is designed to facilitate exploration and analysis of high-throughput data. The platform comes preloaded with logarithmic fold changes from 44 data sets on Mϕ stimulation. We exploited three of these data sets-human primary Mϕs infected with the common lung pathogens Streptococcus pneumoniae, Legionella pneumophila, or Mycobacterium tuberculosis-in a case study to show how our map can be customized with expression data to pinpoint regulated subnetworks and druggable molecules. From the three infection scenarios, we extracted a regulatory core of 41 factors, including TNF, CCL5, CXCL10, IL-18, and IL-12 p40, and identified 140 drugs targeting 16 of them. Our approach promotes a comprehensive systems biology strategy for the exploitation of high-throughput data in the context of Mϕ signal transduction. In conclusion, we provide a set of tools to help scientists unravel details of Mϕ signaling. The interactive version of our Mϕ signal transduction map is accessible online at https://vcells.net/macrophage.

Sphingosine Kinase 1 Regulates Inflammation and Contributes to Acute Lung Injury in Pneumococcal Pneumonia via the Sphingosine-1-Phosphate Receptor 2.

OBJECTIVES: Severe pneumonia may evoke acute lung injury, and sphingosine-1-phosphate is involved in the regulation of vascular permeability and immune responses. However, the role of sphingosine-1-phosphate and the sphingosine-1-phosphate producing sphingosine kinase 1 in pneumonia remains elusive. We examined the role of the sphingosine-1-phosphate system in regulating pulmonary vascular barrier function in bacterial pneumonia. DESIGN: Controlled, in vitro, ex vivo, and in vivo laboratory study. SUBJECTS: Female wild-type and SphK1-deficient mice, 8-10 weeks old. Human postmortem lung tissue, human blood-derived macrophages, and pulmonary microvascular endothelial cells. INTERVENTIONS: Wild-type and SphK1-deficient mice were infected with Streptococcus pneumoniae. Pulmonary sphingosine-1-phosphate levels, messenger RNA expression, and permeability as well as lung morphology were analyzed. Human blood-derived macrophages and human pulmonary microvascular endothelial cells were infected with S. pneumoniae. Transcellular electrical resistance of human pulmonary microvascular endothelial cell monolayers was examined. Further, permeability of murine isolated perfused lungs was determined following exposition to sphingosine-1-phosphate and pneumolysin. MEASUREMENTS AND MAIN RESULTS: Following S. pneumoniae infection, murine pulmonary sphingosine-1-phosphate levels and sphingosine kinase 1 and sphingosine-1-phosphate receptor 2 expression were increased. Pneumonia-induced lung hyperpermeability was reduced in SphK1 mice compared with wild-type mice. Expression of sphingosine kinase 1 in macrophages recruited to inflamed lung areas in pneumonia was observed in murine and human lungs. S. pneumoniae induced the sphingosine kinase 1/sphingosine-1-phosphate system in blood-derived macrophages and enhanced sphingosine-1-phosphate receptor 2 expression in human pulmonary microvascular endothelial cell in vitro. In isolated mouse lungs, pneumolysin-induced hyperpermeability was dose dependently and synergistically increased by sphingosine-1-phosphate. This sphingosine-1-phosphate-induced increase was reduced by inhibition of sphingosine-1-phosphate receptor 2 or its downstream effector Rho-kinase. CONCLUSIONS: Our data suggest that targeting the sphingosine kinase 1-/sphingosine-1-phosphate-/sphingosine-1-phosphate receptor 2-signaling pathway in the lung may provide a novel therapeutic perspective in pneumococcal pneumonia for prevention of acute lung injury.

Legionella pneumophila-Derived Outer Membrane Vesicles Promote Bacterial Replication in Macrophages.

The formation and release of outer membrane vesicles (OMVs) is a phenomenon of Gram-negative bacteria. This includes Legionella pneumophila (L. pneumophila), a causative agent of severe pneumonia. Upon its transmission into the lung, L. pneumophila primarily infects and replicates within macrophages. Here, we analyzed the influence of L. pneumophila OMVs on macrophages. To this end, differentiated THP-1 cells were incubated with increasing doses of Legionella OMVs, leading to a TLR2-dependent classical activation of macrophages with the release of pro-inflammatory cytokines. Inhibition of TLR2 and NF-κB signaling reduced the induction of pro-inflammatory cytokines. Furthermore, treatment of THP-1 cells with OMVs prior to infection reduced replication of L. pneumophila in THP-1 cells. Blocking of TLR2 activation or heat denaturation of OMVs restored bacterial replication in the first 24 h of infection. With prolonged infection-time, OMV pre-treated macrophages became more permissive for bacterial replication than untreated cells and showed increased numbers of Legionella-containing vacuoles and reduced pro-inflammatory cytokine induction. Additionally, miRNA-146a was found to be transcriptionally induced by OMVs and to facilitate bacterial replication. Accordingly, IRAK-1, one of miRNA-146a's targets, showed prolonged activation-dependent degradation, which rendered THP-1 cells more permissive for Legionella replication. In conclusion, L. pneumophila OMVs are initially potent pro-inflammatory stimulators of macrophages, acting via TLR2, IRAK-1, and NF-κB, while at later time points, OMVs facilitate L. pneumophila replication by miR-146a-dependent IRAK-1 suppression. OMVs might thereby promote spreading of L. pneumophila in the host.

MicroRNAs Constitute a Negative Feedback Loop in Streptococcus pneumoniae-Induced Macrophage Activation.

Streptococcus pneumoniae causes high mortality as a major pneumonia-inducing pathogen. In pneumonia, control of innate immunity is necessary to prevent organ damage. We assessed the role of microRNAs (miRNAs) as regulators in pneumococcal infection of human macrophages. Exposure of primary blood-derived human macrophages with pneumococci resulted in transcriptional changes in several gene clusters and a significant deregulation of 10 microRNAs. Computational network analysis retrieved miRNA-146a as one putatively important regulator of pneumococci-induced host cell activation. Its induction depended on bacterial structural integrity and was completely inhibited by blocking Toll-like receptor 2 (TLR-2) or depleting its mediator MyD88. Furthermore, induction of miRNA-146a release did not require the autocrine feedback of interleukin 1ß and tumor necrosis factor α released from infected macrophages, and it repressed the TLR-2 downstream mediators IRAK-1 and TRAF-6, as well as the inflammatory factors cyclooxygenase 2 and interleukin 1ß. In summary, pneumococci recognition induces a negative feedback loop, preventing excessive inflammation via miR-146a and potentially other miRNAs.

Genome-wide Chromatin Profiling of Legionella pneumophila-Infected Human Macrophages Reveals Activation of the Probacterial Host Factor TNFAIP2.

BACKGROUND: Legionella pneumophila is a causative agent of severe pneumonia. Infection leads to a broad host cell response, as evident, for example, on the transcriptional level. Chromatin modifications, which control gene expression, play a central role in the transcriptional response to L. pneumophila METHODS: We infected human-blood-derived macrophages (BDMs) with L. pneumophila and used chromatin immunoprecipitation followed by sequencing to screen for gene promoters with the activating histone 4 acetylation mark. RESULTS: We found the promoter of tumor necrosis factor α-induced protein 2 (TNFAIP2) to be acetylated at histone H4. This factor has not been characterized in the pathology of L. pneumophila TNFAIP2 messenger RNA and protein were upregulated in response to L. pneumophila infection of human-BDMs and human alveolar epithelial (A549) cells. We showed that L. pneumophila-induced TNFAIP2 expression is dependent on the NF-κB transcription factor. Importantly, knock down of TNFAIP2 led to reduced intracellular replication of L. pneumophila Corby in A549 cells. CONCLUSIONS: Taken together, genome-wide chromatin analysis of L. pneumophila-infected macrophages demonstrated induction of TNFAIP2, a NF-κB-dependent factor relevant for bacterial replication.

A mRNA panel for differentiation between acute exacerbation or pneumonia in COPD patients.

Introduction: Patients suffering from chronic obstructive pulmonary disease (COPD) are prone to acute exacerbations (AECOPD) or community acquired pneumonia (CAP), both posing severe risk of morbidity and mortality. There is no available biomarker that correctly separates AECOPD from COPD. However, because CAP and AECOPD differ in aetiology, treatment and prognosis, their discrimination would be important. Methods: This study analysed the ability of selected candidate transcripts from peripheral blood mononuclear cells (PBMCs) to differentiate between patients with AECOPD, COPD & CAP, and CAP without pre-existing COPD. Results: In a previous study, we identified differentially regulated genes between CAP and AECOPD in PBMCs. In the present new cohort, we tested the potential of selected candidate PBMC transcripts to differentiate at early time points AECOPD, CAP+COPD, and CAP without pre-existing COPD. Expression of YWHAG, E2F1 and TDRD9 held predictive power: This gene set predicted diseases markedly better (model accuracy up to 100%) than classical clinical markers like CRP, lymphocyte count and neutrophil count (model accuracy up to 82%). Discussion: In summary, in our cohort expression levels of YWHAG, E2F1 and TDRD9 differentiated with high accuracy between COPD patients suffering from acute exacerbation or CAP.

TLR9- and Src-dependent expression of Krueppel-like factor 4 controls interleukin-10 expression in pneumonia.

The release of potent pro-inflammatory mediators is crucial to mounting an efficient host response during infection. However, excessive inflammation may lead to deleterious tissue damage. This is highlighted in severe pneumococcal pneumonia, in which the delicate balance between a robust inflammatory response necessary to kill pneumococci and the loss of organ function determines the outcome of the disease. We assessed the regulation of the potent anti-inflammatory cytokine interleukin (IL)-10 in pneumococcal infection via Western blot, ELISA and chromatin immunoprecipitation analysis. Streptococcus pneumoniae induced IL-10 expression in mouse lungs and human lung epithelial cells. Pneumococcal infection resulted in a strong induction of Krueppel-like factor (KLF)4 expression in vivo and in vitro. The induction of both IL-10 and KLF4 is mediated by a pathway involving bacterial DNA, Toll-like receptor (TLR)9, MyD88 and Src kinase. KLF4 is recruited to the il10 promoter, and small-interfering RNA-mediated knockdown of KLF4 expression blocked IL-10 expression during pneumococcal infection. In conclusion, KLF4 is induced in a bacterial DNA-TLR9-Src-dependent manner and regulates IL-10 expression, linking the detection of bacterial DNA by TLR9 to the control of an inflammatory response.

Novel protein biomarkers for pneumonia and acute exacerbations in COPD: a pilot study.

Introduction: Community-acquired pneumonia (CAP) and acute exacerbations of chronic obstructive pulmonary disease (AECOPD) result in high morbidity, mortality, and socio-economic burden. The usage of easily accessible biomarkers informing on disease entity, severity, prognosis, and pathophysiological endotypes is limited in clinical practice. Here, we have analyzed selected plasma markers for their value in differential diagnosis and severity grading in a clinical cohort. Methods: A pilot cohort of hospitalized patients suffering from CAP (n = 27), AECOPD (n = 10), and healthy subjects (n = 22) were characterized clinically. Clinical scores (PSI, CURB, CRB65, GOLD I-IV, and GOLD ABCD) were obtained, and interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-2-receptor (IL-2R), lipopolysaccharide-binding protein (LBP), resistin, thrombospondin-1 (TSP-1), lactotransferrin (LTF), neutrophil gelatinase-associated lipocalin (NGAL), neutrophil-elastase-2 (ELA2), hepatocyte growth factor (HGF), soluble Fas (sFas), as well as TNF-related apoptosis-inducing ligand (TRAIL) were measured in plasma. Results: In CAP patients and healthy volunteers, we found significantly different levels of ELA2, HGF, IL-2R, IL-6, IL-8, LBP, resistin, LTF, and TRAIL. The panel of LBP, sFas, and TRAIL could discriminate between uncomplicated and severe CAP. AECOPD patients showed significantly different levels of LTF and TRAIL compared to healthy subjects. Ensemble feature selection revealed that CAP and AECOPD can be discriminated by IL-6, resistin, together with IL-2R. These factors even allow the differentiation between COPD patients suffering from an exacerbation or pneumonia. Discussion: Taken together, we identified immune mediators in patient plasma that provide information on differential diagnosis and disease severity and can therefore serve as biomarkers. Further studies are required for validation in bigger cohorts.

Cell-free biosynthesis combined with deep learning accelerates de novo-development of antimicrobial peptides.

Bioactive peptides are key molecules in health and medicine. Deep learning holds a big promise for the discovery and design of bioactive peptides. Yet, suitable experimental approaches are required to validate candidates in high throughput and at low cost. Here, we established a cell-free protein synthesis (CFPS) pipeline for the rapid and inexpensive production of antimicrobial peptides (AMPs) directly from DNA templates. To validate our platform, we used deep learning to design thousands of AMPs de novo. Using computational methods, we prioritized 500 candidates that we produced and screened with our CFPS pipeline. We identified 30 functional AMPs, which we characterized further through molecular dynamics simulations, antimicrobial activity and toxicity. Notably, six de novo-AMPs feature broad-spectrum activity against multidrug-resistant pathogens and do not develop bacterial resistance. Our work demonstrates the potential of CFPS for high throughput and low-cost production and testing of bioactive peptides within less than 24 h.

NAD+ metabolism is a key modulator of bacterial respiratory epithelial infections.

Lower respiratory tract infections caused by Streptococcus pneumoniae (Spn) are a leading cause of death globally. Here we investigate the bronchial epithelial cellular response to Spn infection on a transcriptomic, proteomic and metabolic level. We found the NAD+ salvage pathway to be dysregulated upon infection in a cell line model, primary human lung tissue and in vivo in rodents, leading to a reduced production of NAD+. Knockdown of NAD+ salvage enzymes (NAMPT, NMNAT1) increased bacterial replication. NAD+ treatment of Spn inhibited its growth while growth of other respiratory pathogens improved. Boosting NAD+ production increased NAD+ levels in immortalized and primary cells and decreased bacterial replication upon infection. NAD+ treatment of Spn dysregulated the bacterial metabolism and reduced intrabacterial ATP. Enhancing the bacterial ATP metabolism abolished the antibacterial effect of NAD+. Thus, we identified the NAD+ salvage pathway as an antibacterial pathway in Spn infections, predicting an antibacterial mechanism of NAD+.

Autoantibodies are highly prevalent in non-SARS-CoV-2 respiratory infections and critical illness.

The widespread presence of autoantibodies in acute infection with SARS-CoV-2 is increasingly recognized, but the prevalence of autoantibodies in non-SARS-CoV-2 infections and critical illness has not yet been reported. We profiled IgG autoantibodies in 267 patients from 5 independent cohorts with non-SARS-CoV-2 viral, bacterial, and noninfectious critical illness. Serum samples were screened using Luminex arrays that included 58 cytokines and 55 autoantigens, many of which are associated with connective tissue diseases (CTDs). Samples positive for anti-cytokine antibodies were tested for receptor blocking activity using cell-based functional assays. Anti-cytokine antibodies were identified in > 50% of patients across all 5 acutely ill cohorts. In critically ill patients, anti-cytokine antibodies were far more common in infected versus uninfected patients. In cell-based functional assays, 11 of 39 samples positive for select anti-cytokine antibodies displayed receptor blocking activity against surface receptors for Type I IFN, GM-CSF, and IL-6. Autoantibodies against CTD-associated autoantigens were also commonly observed, including newly detected antibodies that emerged in longitudinal samples. These findings demonstrate that anti-cytokine and autoantibodies are common across different viral and nonviral infections and range in severity of illness.

Pulmonary inflammatory response and immunomodulation to multiple trauma and hemorrhagic shock in pigs.

BACKGROUND: Patients suffering from severe trauma experience substantial immunological stress. Lung injury is a known risk factor for the development of posttraumatic complications, but information on the long-term course of the pulmonary inflammatory response and treatment with mild hypothermia are scarce. AIM: To investigate the pulmonary inflammatory response to multiple trauma and hemorrhagic shock in a porcine model of combined trauma and to assess the immunomodulatory properties of mild hypothermia. METHODS: Following induction of trauma (blunt chest trauma, liver laceration, tibia fracture), two degrees of hemorrhagic shock (45 and 50%) over 90 (n = 30) and 120 min. (n = 20) were induced. Animals were randomized to hypothermia (33°C) or normothermia (38°C). We evaluated bronchoalveolar lavage (BAL) fluid and tissue levels of cytokines and investigated changes in microRNA- and gene-expression as well as tissue apoptosis. RESULTS: We observed a significant induction of Interleukin (IL) 1ß, IL-6, IL-8, and Cyclooxygenase-2 mRNA in lung tissue. Likewise, an increased IL-6 protein concentration could be detected in BAL-fluid, with a slight decrease of IL-6 protein in animals treated with hypothermia. Lower IL-10 protein levels in normothermia and higher IL-10 protein concentrations in hypothermia accompanied this trend. Tissue apoptosis increased after trauma. However, intervention with hypothermia did not result in a meaningful reduction of pro-inflammatory biomarkers or tissue apoptosis. CONCLUSION: We observed signs of a time-dependent pulmonary inflammation and apoptosis at the site of severe trauma, and to a lower extent in the trauma-distant lung. Intervention with mild hypothermia had no considerable effect during 48 hours following trauma.