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The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus has been causing the COVID-19 pandemic since December 2019, with over 600 million infected persons worldwide and over six million deaths. We investigated the anti-viral effects of polyphenolic green tea ingredients and the synthetic resveratrol analogue 3,3′,4,4′,5,5′-hexahydroxy-trans-stilbene (HHS), a compound with antioxidant, antitumor and anti-HIV properties. In the TCID50 assay, four out of nine green tea constituents showed minor to modest cell protective effects, whereas HHS demonstrated the highest reduction (1103-fold) of the TCID50, indicating pronounced inhibition of virus replication. HHS was also a highly effective inhibitor of SARS-CoV-2 proliferation in VeroE6 cells with an IC50 value of 31.1 µM. HSS also inhibited the binding of the receptor-binding domain (RBD) of the spike protein to the human angiotensin-converting enzyme 2 (ACE2) receptor (RBD-ACE2) binding with 29% at 100 µM and with 9.2% at 50 µM indicating that the SARS-CoV-2 inhibitory effect might at least in part be attributed to the inhibition of virus binding to ACE2. Based on the chemical similarity to other polyphenols, the oral bioavailability of HHS is likely also very low, resulting in blood levels far below the inhibitory concentration of EGCG against SARS-CoV-2 observed in vitro. However, administration of HHS topically as a nose or throat spray would increase concentrations several-fold above the minimal inhibitory concentration (MIC) in the mucosa and might reduce virus load when administered soon after infection. Due to these promising tissue culture results, further preclinical and clinical studies are warranted to develop HHS as an additional treatment option for SARS-CoV-2 infection to complement vaccines, which is and will be the main pillar to combat the COVID-19 pandemic.

Sepsis is a life-threatening condition characterized by uncontrolled systemic inflammation and coagulation, leading to multi-organ failure. Therapeutic options to prevent sepsis-associated immunopathology remain scarce.Here, we established a model of long-lasting disease tolerance during severe sepsis, manifested by diminished immunothrombosis and organ damage in spite of a high pathogen burden. We found that, both neutrophils and B cells emerged as key regulators of tissue integrity. Enduring changes in the transcriptional profile of neutrophils, included upregulated Cxcr4 expression in protected, tolerant hosts. Neutrophil Cxcr4 upregulation required the presence of B cells, suggesting that B cells promoted tissue tolerance by suppressing tissue damaging properties of neutrophils. Finally, therapeutic administration of a Cxcr4 agonist successfully promoted tissue tolerance and prevented liver damage during sepsis. Our findings highlight the importance of a critical B-cell/neutrophil interaction during sepsis and establish neutrophil Cxcr4 activation as a potential means to promote disease tolerance during sepsis.SummaryWe show that a B cell/neutrophil interaction in the bone marrow facilitates tissue tolerance during severe sepsis. By affecting neutrophil Cxcr4 expression, B cells can impact neutrophil effector functions. Finally, therapeutic activation of Cxcr4 successfully promoted tissue tolerance and prevented liver damage during sepsis.

Despite tremendous progress in the understanding of COVID-19, mechanistic insight into immunological, disease-driving factors remains limited. We generated maVie16, a mouse-adapted SARS-CoV-2, by serial passaging of a human isolate. In silico modeling revealed how only three Spike mutations of maVie16 enhanced interaction with murine ACE2. maVie16 induced profound pathology in BALB/c and C57BL/6 mice, and the resulting mouse COVID-19 (mCOVID-19) replicated critical aspects of human disease, including early lymphopenia, pulmonary immune cell infiltration, pneumonia, and specific adaptive immunity. Inhibition of the proinflammatory cytokines IFNγ and TNF substantially reduced immunopathology. Importantly, genetic ACE2-deficiency completely prevented mCOVID-19 development. Finally, inhalation therapy with recombinant ACE2 fully protected mice from mCOVID-19, revealing a novel and efficient treatment. Thus, we here present maVie16 as a new tool to model COVID-19 for the discovery of new therapies and show that disease severity is determined by cytokine-driven immunopathology and critically dependent on ACE2 in vivo.

To develop a universal strategy to block SARS-CoV-2 cellular entry and infection represents a central aim for effective COVID-19 therapy. The growing impact of emerging variants of concern increases the urgency for development of effective interventions. Since ACE2 is the critical SARS-CoV-2 receptor and all tested variants bind to ACE2, some even at much increased affinity (see accompanying paper), we hypothesized that aerosol administration of clinical grade soluble human recombinant ACE2 (APN01) will neutralize SARS-CoV-2 in the airways, limit spread of infection in the lung and mitigate lung damage caused by deregulated signaling in the renin-angiotensin (RAS) and Kinin pathways. Here we show that intranasal administration of APN01 in a mouse model of SARS-CoV-2 infection dramatically reduced weight loss and prevented animal death. As a prerequisite to a clinical trial, we evaluated both virus binding activity and enzymatic activity for cleavage of Ang II following aerosolization. We report successful aerosolization for APN01, retaining viral binding as well as catalytic RAS activity. Dose range-finding and IND-enabling repeat-dose aerosol toxicology testing were conducted in dogs. Twice daily aerosol administration for two weeks at the maximum feasible concentration revealed no notable toxicities. Based on these results, a Phase I clinical trial in healthy volunteers can now be initiated, with subsequent Phase II testing in individuals with SARS-CoV-2 infection. This strategy could be used to develop a viable and rapidly actionable therapy to prevent and treat COVID-19, against all current and future SARS-CoV-2 variants., One Sentence Summary: Preclinical development and evaluation of aerosolized soluble recombinant human ACE2 (APN01) administered as a COVID-19 intervention is reported.

SummaryDespite tremendous progress in the understanding of COVID-19, mechanistic insight into immunological, disease-driving factors remains limited. We generated maVie16, a mouse-adapted SARS-CoV-2, by serial passaging of a human isolate. In silico modelling revealed how Spike mutations of maVie16 enhanced interaction with murine ACE2. MaVie16 induced profound pathology in BALB/c and C57BL/6 mice and the resulting mouse COVID-19 (mCOVID-19) replicated critical aspects of human disease, including early lymphopenia, pulmonary immune cell infiltration, pneumonia and specific adaptive immunity. Inhibition of the proinflammatory cytokines IFNγ and TNF substantially reduced immunopathology. Importantly, genetic ACE2-deficiency completely prevented mCOVID-19 development. Finally, inhalation therapy with recombinant ACE2 fully protected mice from mCOVID-19, revealing a novel and efficient treatment. Thus, we here present maVie16 as a new tool to model COVID-19 for the discovery of new therapies and show that disease severity is determined by cytokine-driven immunopathology and critically dependent on ACE2 in vivo.Key pointsThe mouse-adapted SARS-CoV-2 strain maVie16 causes fatal disease in BALB/c mice and substantial inflammation, pneumonia and immunity in C57BL/6 miceTNFα/IFNγ blockade ameliorates maVie16-induced immunopathologyMaVie16 infection depends on ACE2 and soluble ACE2 inhalation can prevent disease

Purpose Allograft adaptation after lung transplantation is a multi-factor process. We hypothesized that early adaptation can influence the risk of matrix remodeling and CLAD development by shaping the inflammatory state of the lung environment. In a longitudinal study, we followed a defined patient cohort in year 1 p.t. and detected ordered cell composition changes with repercussions in the microbiome, small molecular composition and lipidome in BAL samples. We delimited clusters of pro-inflammatory factors and identified long-chain lipid species from the lung environment that modulated inflammatory response in vitro. Methods We used FACS to identify cell populations and 16S sequencing, metabolomics, lipidomics to characterize the lung environment. With correlations and hierarchical clustering we extracted pro- and anti-inflammatory clusters around well-characterized cell populations. For detection of inflammation drivers, we quantified feature differential abundances in samples with more or less neutrophilia. Predicted candidates were tested to modify inflammatory responses in vitro by screening IL-6 production of LPS-triggered macrophages. Results Early after surgery, the allograft experienced neutrophilia, a lack of resident macrophages and a gradual influx of host-derived macrophage-precursors directly resonating with changes in the pulmonary microenvironment. The computational analysis revealed features that clustered either with neutrophils or alveolar macrophages. From these, 30% showed significant abundance differences in high and low neutrophilia conditions. Consistently, certain ceramide species displayed antagonistic behavior to several phosphatidylcholines. We tested their capability of modifying macrophage response and found the ceramide to enhance, and PCs to reduce IL-6 production. Conclusion Our study suggests that the lung microenvironment plays a key role in allograft adaptation and specific lipid species are capable of modulating cellular inflammatory response in a differentiated manner early after transplantation. Our results provide first fundamental concepts for developing concrete precision therapies based on reprogramming tissue homoeostasis and inflammation management.

RationaleLung transplantation is the ultimate treatment option for patients with end-stage respiratory diseases but bears the highest mortality rate among all solid organ transplantations due to chronic lung allograft dysfunction (CLAD). The mechanisms leading to CLAD remain elusive due to an insufficient understanding of the complex post-transplant adaptation processes.ObjectivesTo better understand these lung adaptation processes after transplantation and to investigate their association with future changes in allograft function.MethodsWe performed an exploratory cohort study of bronchoalveolar lavage samples from 78 lung recipients and donors. We analysed the alveolar microbiome using 16S rRNA sequencing, the cellular composition using flow cytometry, as well as metabolome and lipidome profiling.Measurements and main resultsWe established distinct temporal dynamics for each of the analysed data sets. Comparing matched donor and recipient samples, we revealed that recipient-specific as well as environmental factors, rather than the donor microbiome, shape the long-term lung microbiome. We further discovered that the abundance of certain bacterial strains correlated with underlying lung diseases even after transplantation. A decline in forced expiratory volume during the first second (FEV1) is a major characteristic of lung allograft dysfunction in transplant recipients. By using a machine learning approach, we could accurately predict future changes in FEV1 from our multi-omics data, whereby microbial profiles showed a particularly high predictive power.ConclusionBronchoalveolar microbiome, cellular composition, metabolome and lipidome show specific temporal dynamics after lung transplantation. The lung microbiome can predict future changes in lung function with high precision.

Cytomegalovirus (CMV) has a high prevalence worldwide, is often fatal for immunocompromised patients, and causes bone marrow suppression. Deficiency of signal transducer and activator of transcription 1 (STAT1) results in severely impaired antiviral immunity. We have used cell- type restricted deletion of Stat1 to determine the importance of myeloid cell activity for the defense against murine CMV (MCMV). We show that myeloid STAT1 limits MCMV burden and infection- associated pathology in the spleen but does not affect ultimate clearance of infection. Unexpectedly, we found an essential role of myeloid STAT1 in the induction of extramedullary hematopoiesis (EMH). The EMH- promoting function of STAT1 was not restricted to MCMV infection but was also observed during CpG oligodeoxynucleotide-induced sterile inflammation. Collectively, we provide genetic evidence that signaling through STAT1 in myeloid cells is required to restrict MCMV at early time points post-infection and to induce compensatory hematopoiesis in the spleen.

Purpose Spirometric lung function is one of the central clinical parameters to assess allograft function after lung transplantation (LTX). Reduction in FEV1 can be the result of infections, rejections episodes and most importantly development of CLAD. Currently, there is no means to predict short-term changes in lung function after LTX. Methods Bronchoalveolar samples of patients after standard double LTX were taken during follow-up bronchoscopy at multiple different time points. Lipidomic, metabolomic, flow cytometric and bacterial 16S rRNA gene sequencing (microbiome) data were analyzed. We then used a machine learning approach to predict future changes in FEV1 from these sample data to characterize patient lung function trajectories. We trained support vector machine (SVM) regressors on the collected lipidomic, metabolomic, microbiome and flow cytometry datasets. Changes in FEV1 within 30, 60 or 90 days after lavage sample collection were used as response variables. To train hyper-parameters and evaluate model accuracy, a nested leave-one-out cross validation scheme was used. Model accuracy was benchmarked against a model trained on clinical metadata. Results At a prediction timeframe of 30 days, lipidomic data were available from 34 samples of 20 patients. Lipidomics showed the highest predictive power for short-term changes 30 and 60 days after sample collection. Prediction accuracy (R2) of intra-alveolar lipid composition for a 30-day projection was 0.24, meaning that BAL lipid profiles could explain more than 20 percent of total variation in relative change in FEV1 for this time span. R² for clinical metadata alone was only 0.1, and metabolomics, microbiome and FACS analysis of BAL showed no predictive accuracy at this time point. Conclusion Our results suggest that the intra-alveolar lipid composition is a powerful predictor of short-term changes in lung allograft function. This could potentially facilitate pre-emptive therapeutic interventions.

Purpose The pulmonary microbiome after lung transplantation has recently come into the focus of research. Especially, host-microbiome interactions are thought to be an important factor in graft-related immunological processes. In lung transplantation, temporal changes of the pulmonary microbiome have not yet been elucidated. Methods In a total cohort of 80 patients receiving standard double lung transplantation, 50 bronchioalveolar lavage (BAL) samples from donors were collected prior to cold ischemia. After transplantation, 128 BAL samples were collected at various time points of routine follow-up bronchoscopies between 0 and 400 days post-transplant. Bacterial 16S rRNA gene sequencing was conducted to analyze the composition of the pulmonary microbiome in these samples. Results The lung microbiome showed significant temporal dynamics after lung transplantation. Recipient-donor similarity between matched samples decreased after the first week post transplantation. Underlying transplant indications were significantly associated with microbial profiles even after transplantation. Shannon diversity and Chao1 richness at the species level showed an increasing diversity of the microbiome over time. Conclusion Our data provides new insights into the dynamics of microbial profiles after transplantation. We observed that recipient-associated factors, rather than the donor microbiome, shape the lung microbiome after transplantation.

Red blood cells (RBCs) comprise 84% of our body's cells and have the essential function of transporting oxygen from the lungs to all organs. The daily production rate of RBCs is enormous and the pathways mediating this process are quite complex. One important aspect hereby is the synthesis of hemoglobin and heme, the iron-containing prosthetic group that enables hemoglobin to bind oxygen.RBCs differentiate within the erythropoietic niche, which consists of erythroid precursors and specialized macrophages. These so-called nursing macrophages provide nutrients and iron, a fundamental component of heme. The precise mechanisms of heme transport within a cell and the potential of heme transfer between cells is not completely understood. To get insights into heme transport we took advantage of the cytotoxic capacity of heme and performed a CRISPR-Cas9 loss of function screen by focusing on SLC transporters. We identified SLC20A1 as an essential protein mediating heme toxicity and verified that intracellular heme levels as well as heme-induced downstream gene inductions were reduced in the absence of Slc20a1. Based on these evidences we hypothesized that SLC20A1 might be involved in the trafficking of heme, possibly important during erythropoiesis, since mice lacking Slc20a1 exhibit embryonic lethality due to liver apoptosis and anemia (Beck L. et al., PLoS One 2013). To test the biological role of Slc20a1 in adult mice, we conditionally deleted Slc20a1 using tamoxifen in ERT2-Cre Slc20a1fl animals and observed the spontaneous development of severe anemia and splenomegaly within 2 weeks after tamoxifen administration. We further discovered that the anemia-related expansion of red pulp macrophages (RPM) consisted predominantly of non-recombined wild type cells, whereas successfully recombined (Slc20a1 deficient) cells seemed stuck in the monocytic stage. These data suggest that Slc20a1 might be involved in the differentiation and maturation of monocytes to RPMs. When we performed a specific Slc20a1 deletion in nursing macrophages (CD169 Cre), mice neither showed signs of anemia, nor experienced impaired recovery upon anemia induction following phlebotomy or induction of hemolysis. However, deleting Sc20a1 specifically in the erythroid compartment using Slc20a1fl mice crossed to erythropoietin receptor-cre (EpoR-Cre) animals, resulted in embryonic lethality. Mice died around E12.5 due to severe anemia. Analysis of E11.5 animals disclosed erythroid precursors to be arrested in the pro-erythroblast stage.These data suggest that SLC20A1 is a protein involved in heme-mediated toxicity and possibly in the trafficking of heme with strong impact on fetal and adult erythropoiesis. Disclosures No relevant conflicts of interest to declare.

Cytomegalovirus (CMV) has a high prevalence worldwide, is often fatal for immunocompromised patients and causes bone marrow suppression. Deficiency for signal transducer and activator of transcription 1 (STAT1) results in severely impaired antiviral immunity. We have used cell-type restricted deletion of Stat1 to determine the importance of macrophage activity for the defence against murine CMV (MCMV). We show that macrophage STAT1 limits MCMV burden and infection-associated pathology in the spleen, but does not impact on ultimate clearance of infection. Surprisingly, we found an essential role of macrophage STAT1 in the induction of extramedullary hematopoiesis (EMH). The EMH-promoting function of STAT1 was not only observed after MCMV infection but also during CpG oligodeoxynucleotide-induced sterile inflammation. Collectively, we provide the first genetic evidence that signalling through STAT1 in macrophages is required to restrict MCMV at early time points post infection and to induce compensatory hematopoiesis in the spleen.

During infections, TLR-mediated responses require tight regulation to allow for pathogen removal, while preventing overwhelming inflammation and immunopathology. The triggering receptor expressed on myeloid cells (TREM)-2 negatively regulates inflammation by macrophages and impacts on phagocytosis, but the function of endogenous TREM-2 during infections is poorly understood. We investigated TREM-2's role in regulating TLR4-mediated inflammation by studying wild-type and TREM-2(-/-) mice challenged with LPS and found TREM-2 to dampen early inflammation. Augmented early inflammation in TREM-2(-/-) animals was followed by an accelerated resolution and ultimately improved survival, associated with the induction of the negative regulator A20. Upon infection with Escherichia coli, the otherwise beneficial effect of an exaggerated early immune response in TREM-2(-/-) animals was counteracted by a 50% reduction in bacterial phagocytosis. In line with this, TREM-2(-/-) peritoneal macrophages (PMs) exhibited augmented inflammation following TLR4 stimulation, demonstrating the presence and negative regulatory functionality of TREM-2 on primary PMs. Significantly, we identified a high turnover rate because TREM-2 RNA is 25-fold down-regulated and the protein proteasomally degraded upon LPS encounter, thus ensuring a tightly regulated and versatile system that modulates inflammation. Our results illustrate TREM-2's effects on infection-triggered inflammation and identify TREM-2 as a potential target to prevent overwhelming inflammation while preserving antibacterial-effector functions.

Lipid metabolism is increasingly being appreciated to affect immunoregulation, inflammation and pathology. In this study we found that mice infected with lymphocytic choriomeningitis virus (LCMV) exhibit global perturbations of circulating serum lipids. Mice lacking the lipid-sensing surface receptor triggering receptor expressed on myeloid cells 2 (Trem2 −/−) were protected from LCMV-induced hepatitis and showed improved virus control despite comparable virus-specific T cell responses. Non-hematopoietic expression of TREM2 was found to be responsible for aggravated hepatitis, indicating a novel role for TREM2 in the non-myeloid compartment. These results suggest a link between virus-perturbed lipids and TREM2 that modulates liver pathogenesis upon viral infection. Targeted interventions of this immunoregulatory axis may ameliorate tissue pathology in hepatitis.

International audience; Hemolysis drives susceptibility to bacterial infections and predicts poor outcome from sepsis. These detrimental effects are commonly considered to be a consequence of heme-iron serving as a nutrient for bacteria. We employed a Gram-negative sepsis model and found that elevated heme levels impaired the control of bacterial proliferation independently of heme-iron acquisition by pathogens. Heme strongly inhibited phagocytosis and the migration of human and mouse phagocytes by disrupting actin cytoskeletal dynamics via activation of the GTP-binding Rho family protein Cdc42 by the guanine nucleotide exchange factor DOCK8. A chemical screening approach revealed that quinine effectively prevented heme effects on the cytoskeleton, restored phagocytosis and improved survival in sepsis. These mechanistic insights provide potential therapeutic targets for patients with sepsis or hemolytic disorders.

Summary Lipid metabolism and receptor-mediated signaling are highly intertwined processes that cooperate to fulfill cellular functions and safeguard cellular homeostasis. Activation of Toll-like receptors (TLRs) leads to a complex cellular response, orchestrating a diverse range of inflammatory events that need to be tightly controlled. Here, we identified the GPI-anchored Sphingomyelin Phosphodiesterase, Acid-Like 3B (SMPDL3B) in a mass spectrometry screening campaign for membrane proteins co-purifying with TLRs. Deficiency of Smpdl3b in macrophages enhanced responsiveness to TLR stimulation and profoundly changed the cellular lipid composition and membrane fluidity. Increased cellular responses could be reverted by re-introducing affected ceramides, functionally linking membrane lipid composition and innate immune signaling. Finally, Smpdl3b-deficient mice displayed an intensified inflammatory response in TLR-dependent peritonitis models, establishing its negative regulatory role in vivo. Taken together, our results identify the membrane-modulating enzyme SMPDL3B as a negative regulator of TLR signaling that functions at the interface of membrane biology and innate immunity., Graphical Abstract, Highlights • Identification of SMPDL3B as lipid-modulating phosphodiesterase on macrophages • Negative regulatory role for SMPDL3B in Toll-like receptor function • Strong influence of SMPDL3B on membrane lipid composition and fluidity • Smpdl3b-deficient mice show enhanced responsiveness in TLR-dependent peritonitis, Heinz et al. identify the lipid-modulating phosphodiesterase SMPDL3B as negative regulator of Toll-like receptor function. Smpdl3b-deficiency strongly affected macrophage lipid composition and fluidity and led to higher responsiveness to TLR stimulation. Peritonitis models in Smpdl3b-deficient mice confirmed the negative regulatory role in vivo.

Clearance of invading pathogens is essential to preventing overwhelming inflammation and sepsis that are symptomatic of bacterial peritonitis. Macrophages participate in this innate immune response by engulfing and digesting pathogens, a process called phagocytosis. Oxidized phospholipids (OxPL) are danger-associated molecular patterns (DAMPs) generated in response to infection that can prevent the phagocytic clearance of bacteria. We investigated the mechanism underlying OxPL action in macrophages. Exposure to OxPL induced alterations in actin polymerization, resulting in spreading of peritoneal macrophages and diminished uptake of E. coli. Pharmacological and cell-based studies showed that an anchored pool of PKA mediates the effects of OxPL. Gene silencing approaches identified the A-kinase anchoring protein (AKAP) WAVE1 as an effector of OxPL action in vitro. Chimeric Wave1(-/-) mice survived significantly longer after infection with E. coli and OxPL treatment in vivo. Moreover, we found that endogenously generated OxPL in human peritoneal dialysis fluid from end-stage renal failure patients inhibited phagocytosis via WAVE1. Collectively, these data uncover an unanticipated role for WAVE1 as a critical modulator of the innate immune response to severe bacterial infections.

Macrophages play a key role in responding to pathogens and initiate an inflammatory response to combat microbe multiplication. Deactivation of macrophages facilitates resolution of the inflammatory response. Deactivated macrophages are characterized by an immunosuppressive phenotype, but the lack of unique markers that can reliably identify these cells explains the poorly defined biological role of this macrophage subset. We identified lipocalin 2 (LCN2) as both a marker of deactivated macrophages and a macrophage deactivator. We show that LCN2 attenuated the early inflammatory response and impaired bacterial clearance, leading to impaired survival of mice suffering from pneumococcal pneumonia. LCN2 induced IL-10 formation by macrophages, skewing macrophage polarization in a STAT3-dependent manner. Pulmonary LCN2 levels were tremendously elevated during bacterial pneumonia in humans, and high LCN2 levels were indicative of a detrimental outcome from pneumonia with Gram-positive bacteria. Our data emphasize the importance of macrophage deactivation for the outcome of pneumococcal infections and highlight the role of LCN2 and IL-10 as determinants of macrophage performance in the respiratory tract.