ABSTRACT
Background: Toll-like receptors (TLRs) play a pivotal role in the immunologic response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Exaggerated inflammatory response of innate immune cells, however, may drive morbidity and death in Coronavirus disease 19 (COVID-19). Objective: We investigated the engagement of SARS-CoV-2 with TLR4 in order to better understand how to tackle hyperinflammation in COVID-19. Methods: We combined RNA-sequencing data of human lung tissue and of bronchoalveolar lavage fluid cells derived from COVID-19 patients with functional studies in human macrophages using SARS-CoV-2 spike proteins and viable SARS-CoV-2. Pharmacological inhibitors as well as gene editing with CRISPR/Cas9 were used to delineate the signalling pathways involved. Results: We found TLR4 to be the most abundantly upregulated TLR in human lung tissue irrespective of the underlying pathology. Accordingly, bronchoalveolar lavage fluid cells from patients with severe COVID-19 showed an NF-κB-pathway dominated immune response, whereas they were mostly defined by type I interferon signalling in moderate COVID-19. Mechanistically, we found the Spike ectodomain, but not receptor binding domain monomer to induce TLR4-dependent inflammation in human macrophages. By using pharmacological inhibitors as well as CRISPR/Cas9 deleted macrophages, we identify SARS-CoV-2 to engage canonical TLR4-MyD88 signalling. Importantly, we demonstrate that TLR4 blockage prevents exaggerated inflammatory responses in human macrophages infected with different SARS-CoV-2 variants, including immune escape variants B.1.1.7.-E484K and B.1.1.529 (omicron). Conclusion: Our study critically extends the current knowledge on TLR-mediated hyperinflammatory responses to SARS-CoV-2 in human macrophages, paving the way for novel approaches to tackle severe COVID-19. Take-home message: Our study combining human lung transcriptomics with functional studies in human macrophages clearly supports the design and development of TLR4 - directed therapeutics to mitigate hyperinflammation in severe COVID-19.
ABSTRACT
Background: Klebsiella pneumoniae (KP) is a major cause of hospital-acquired infections, such as pneumonia. Moreover, it is classified as a pathogen of concern due to sprawling anti-microbial resistance. During infection, the gram-negative pathogen is capable of establishing an intracellular niche in macrophages by altering cellular metabolism. One factor critically affecting the host-pathogen interaction is the availability of essential nutrients, like iron, which is required for KP to proliferate but which also modulates anti-microbial immune effector pathways. We hypothesized, that KP manipulates macrophage iron homeostasis to acquire this crucial nutrient for sustained proliferation. Methods: We applied an in-vitro infection model, in which human macrophage-like PMA-differentiated THP1 cells were infected with KP (strain ATCC 43816). During a 24-h course of infection, we quantified the number of intracellular bacteria via serial plating of cell lysates and evaluated the effects of different stimuli on intracellular bacterial numbers and iron acquisition. Furthermore, we analyzed host and pathogen specific gene and protein expression of key iron metabolism molecules. Results: Viable bacteria are recovered from macrophage cell lysates during the course of infection, indicative of persistence of bacteria within host cells and inefficient pathogen clearing by macrophages. Strikingly, following KP infection macrophages strongly induce the expression of the main cellular iron importer transferrin-receptor-1 (TFR1). Accordingly, intracellular KP proliferation is further augmented by the addition of iron loaded transferrin. The induction of TFR1 is mediated via the STAT-6-IL-10 axis, and pharmacological inhibition of this pathway reduces macrophage iron uptake, elicits bacterial iron starvation, and decreases bacterial survival. Conclusion: Our results suggest, that KP manipulates macrophage iron metabolism to acquire iron once confined inside the host cell and enforces intracellular bacterial persistence. This is facilitated by microbial mediated induction of TFR1 via the STAT-6-IL-10 axis. Mechanistic insights into immune metabolism will provide opportunities for the development of novel antimicrobial therapies.
ABSTRACT
BACKGROUND: Calcific aortic valve disease (CAVD) is characterized by a phenotypic switch of valvular interstitial cells to bone-forming cells. Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors at the interface between innate immunity and tissue repair. Type I interferons (IFNs) are not only crucial for an adequate antiviral response but also implicated in bone formation. We hypothesized that the accumulation of endogenous TLR3 ligands in the valvular leaflets may promote the generation of osteoblast-like cells through enhanced type I IFN signaling. METHODS: Human valvular interstitial cells isolated from aortic valves were challenged with mechanical strain or synthetic TLR3 agonists and analyzed for bone formation, gene expression profiles, and IFN signaling pathways. Different inhibitors were used to delineate the engaged signaling pathways. Moreover, we screened a variety of potential lipids and proteoglycans known to accumulate in CAVD lesions as potential TLR3 ligands. Ligand-receptor interactions were characterized by in silico modeling and verified through immunoprecipitation experiments. Biglycan (Bgn), Tlr3, and IFN-α/ß receptor alpha chain (Ifnar1)-deficient mice and a specific zebrafish model were used to study the implication of the biglycan (BGN)-TLR3-IFN axis in both CAVD and bone formation in vivo. Two large-scale cohorts (GERA [Genetic Epidemiology Research on Adult Health and Aging], n=55 192 with 3469 aortic stenosis cases; UK Biobank, n=257 231 with 2213 aortic stenosis cases) were examined for genetic variation at genes implicated in BGN-TLR3-IFN signaling associating with CAVD in humans. RESULTS: Here, we identify TLR3 as a central molecular regulator of calcification in valvular interstitial cells and unravel BGN as a new endogenous agonist of TLR3. Posttranslational BGN maturation by xylosyltransferase 1 (XYLT1) is required for TLR3 activation. Moreover, BGN induces the transdifferentiation of valvular interstitial cells into bone-forming osteoblasts through the TLR3-dependent induction of type I IFNs. It is intriguing that Bgn-/-, Tlr3-/-, and Ifnar1-/- mice are protected against CAVD and display impaired bone formation. Meta-analysis of 2 large-scale cohorts with >300 000 individuals reveals that genetic variation at loci relevant to the XYLT1-BGN-TLR3-interferon-α/ß receptor alpha chain (IFNAR) 1 pathway is associated with CAVD in humans. CONCLUSIONS: This study identifies the BGN-TLR3-IFNAR1 axis as an evolutionarily conserved pathway governing calcification of the aortic valve and reveals a potential therapeutic target to prevent CAVD.
Subject(s)
Aortic Valve Stenosis , Calcinosis , Adult , Animals , Humans , Mice , Aortic Valve/pathology , Aortic Valve Stenosis/pathology , Biglycan/metabolism , Calcinosis/metabolism , Cells, Cultured , Toll-Like Receptor 3/genetics , Toll-Like Receptor 3/metabolism , ZebrafishABSTRACT
Anemia is a major health issue and associated with increased morbidity. Iron deficiency anemia (IDA) is the most prevalent, followed by anemia of chronic disease (ACD). IDA and ACD often co-exist, challenging diagnosis and treatment. While iron supplementation is the first-line therapy for IDA, its optimal route of administration and the efficacy of different repletion strategies in ACD are elusive. Female Lewis rats were injected with group A streptococcal peptidoglycan-polysaccharide (PG-APS) to induce inflammatory arthritis with associated ACD and/or repeatedly phlebotomized and fed with a low iron diet to induce IDA, or a combination thereof (ACD/IDA). Iron was either supplemented by daily oral gavage of ferric maltol or by weekly intravenous (i.v.) injection of ferric carboxymaltose for up to 4 weeks. While both strategies reversed IDA, they remained ineffective to improve hemoglobin (Hb) levels in ACD, although oral iron showed slight amelioration of various erythropoiesis-associated parameters. In contrast, both iron treatments significantly increased Hb in ACD/IDA. In ACD and ACD/IDA animals, i.v. iron administration resulted in iron trapping in liver and splenic macrophages, induction of ferritin expression and increased circulating levels of the iron hormone hepcidin and the inflammatory cytokine interleukin-6, while oral iron supplementation reduced interleukin-6 levels. Thus, oral and i.v. iron resulted in divergent effects on systemic and tissue iron homeostasis and inflammation. Our results indicate that both iron supplements improve Hb in ACD/IDA, but are ineffective in ACD with pronounced inflammation, and that under the latter condition, i.v. iron is trapped in macrophages and may enhance inflammation.
Subject(s)
Anemia, Iron-Deficiency , Anemia , Female , Animals , Rats , Interleukin-6 , Rats, Inbred Lew , Anemia/diagnosis , Iron/metabolism , Anemia, Iron-Deficiency/drug therapy , Anemia, Iron-Deficiency/etiology , Anemia, Iron-Deficiency/diagnosis , Inflammation/drug therapyABSTRACT
The sequestration of iron in case of infection, termed nutritional immunity, is an established strategy of host defense. However, the interaction between pathogens and the mammalian iron storage protein ferritin is hitherto not completely understood. To better characterize the function of ferritin in Gram-negative infections, we incubated iron-starved cultures of Salmonella Typhimurium and knockout mutant strains defective for major iron uptake pathways or Escherichia coli with horse spleen ferritin or ionic iron as the sole iron source. Additionally, we added bovine superoxide dismutase and protease inhibitors to the growth medium to assess the effect of superoxide and bacterial proteases, respectively, on Salmonella proliferation and reductive iron release. Compared to free ionic iron, ferritin-bound iron was less available to Salmonella, but was still sufficient to significantly enhance the growth of the bacteria. In the absence of various iron acquisition genes, the availability of ferritin iron further decreased. Supplementation with superoxide dismutase significantly reduced the growth of the ΔentC knockout strain with holoferritin as the sole iron source in comparison with ionic ferrous iron. In contrast, this difference was not observed in the wildtype strain, suggesting that superoxide dismutase undermines bacterial iron uptake from ferritin by siderophore-independent mechanisms. Ferritin seems to diminish iron availability for bacteria in comparison to ionic iron, and its iron sequestering effect could possibly be enhanced by host superoxide dismutase activity.
Subject(s)
Ferritins , Iron , Cattle , Animals , Horses , Ferritins/metabolism , Iron/metabolism , Enterobacteriaceae , Salmonella typhimurium , Superoxide Dismutase/metabolism , Escherichia coli/metabolism , Mammals/metabolismABSTRACT
Background: Pneumonia is often elicited by bacteria and can be associated with a severe clinical course, respiratory failure and the need for mechanical ventilation. In the alveolus, type-2-alveolar-epithelial-cells (AECII) contribute to innate immune functions. We hypothesized that AECII actively adapt cellular iron homeostasis to restrict this essential nutrient from invading pathogens - a defense strategy termed 'nutritional immunity', hitherto mainly demonstrated for myeloid cells. Methods: We established an in-vitro infection model using the human AECII-like cell line A549. We infected cells with Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli), two gram-negative bacteria with different modes of infection and frequent causes of hospital-acquired pneumonia. We followed the entry and intracellular growth of these gram-negative bacteria and analyzed differential gene expression and protein levels of key inflammatory and iron metabolism molecules. Results: Both, K. pneumoniae and E. coli are able to invade A549 cells, whereas only K. pneumoniae is capable of proliferating intracellularly. After peak bacterial burden, the number of intracellular pathogens declines, suggesting that epithelial cells initiate antimicrobial immune effector pathways to combat bacterial proliferation. The extracellular pathogen E. coli induces an iron retention phenotype in A549 cells, mainly characterized by the downregulation of the pivotal iron exporter ferroportin, the upregulation of the iron importer transferrin-receptor-1 and corresponding induction of the iron storage protein ferritin. In contrast, cells infected with the facultative intracellular bacterium K. pneumoniae exhibit an iron export phenotype indicated by ferroportin upregulation. This differential regulation of iron homeostasis and the pathogen-specific inflammatory reaction is likely mediated by oxidative stress. Conclusion: AECII-derived A549 cells show pathogen-specific innate immune functions and adapt their iron handling in response to infection. The differential regulation of iron transporters depends on the preferential intra- or extracellular localization of the pathogen and likely aims at limiting bacterial iron availability.
Subject(s)
Escherichia coli Infections , Klebsiella Infections , Pneumonia , Epithelial Cells , Escherichia coli/metabolism , Gram-Negative Bacteria/metabolism , Humans , Iron/metabolism , Klebsiella Infections/microbiology , Klebsiella pneumoniae/metabolismABSTRACT
Iron is an essential component for metabolic processes, including oxygen transport within hemoglobin, tricarboxylic acid (TCA) cycle activity, and mitochondrial energy transformation. Iron deficiency can thus lead to metabolic dysfunction and eventually result in iron deficiency anemia (IDA), which affects approximately 1.5 billion people worldwide. Using a rat model of IDA induced by phlebotomy, we studied the effects of IDA on mitochondrial respiration in peripheral blood mononuclear cells (PBMCs) and the liver. Furthermore, we evaluated whether the mitochondrial function evaluated by high-resolution respirometry in PBMCs reflects corresponding alterations in the liver. Surprisingly, mitochondrial respiratory capacity was increased in PBMCs from rats with IDA compared to the controls. In contrast, mitochondrial respiration remained unaffected in livers from IDA rats. Of note, citrate synthase activity indicated an increased mitochondrial density in PBMCs, whereas it remained unchanged in the liver, partly explaining the different responses of mitochondrial respiration in PBMCs and the liver. Taken together, these results indicate that mitochondrial function determined in PBMCs cannot serve as a valid surrogate for respiration in the liver. Metabolic adaptions to iron deficiency resulted in different metabolic reprogramming in the blood cells and liver tissue.
ABSTRACT
BACKGROUND & AIMS: Crohn's disease (CD) globally emerges with Westernization of lifestyle and nutritional habits. However, a specific dietary constituent that comprehensively evokes gut inflammation in human inflammatory bowel diseases remains elusive. We aimed to delineate how increased intake of polyunsaturated fatty acids (PUFAs) in a Western diet, known to impart risk for developing CD, affects gut inflammation and disease course. We hypothesized that the unfolded protein response and antioxidative activity of glutathione peroxidase 4 (GPX4), which are compromised in human CD epithelium, compensates for metabolic perturbation evoked by dietary PUFAs. METHODS: We phenotyped and mechanistically dissected enteritis evoked by a PUFA-enriched Western diet in 2 mouse models exhibiting endoplasmic reticulum (ER) stress consequent to intestinal epithelial cell (IEC)-specific deletion of X-box binding protein 1 (Xbp1) or Gpx4. We translated the findings to human CD epithelial organoids and correlated PUFA intake, as estimated by a dietary questionnaire or stool metabolomics, with clinical disease course in 2 independent CD cohorts. RESULTS: PUFA excess in a Western diet potently induced ER stress, driving enteritis in Xbp1-/-IEC and Gpx4+/-IEC mice. ω-3 and ω-6 PUFAs activated the epithelial endoplasmic reticulum sensor inositol-requiring enzyme 1α (IRE1α) by toll-like receptor 2 (TLR2) sensing of oxidation-specific epitopes. TLR2-controlled IRE1α activity governed PUFA-induced chemokine production and enteritis. In active human CD, ω-3 and ω-6 PUFAs instigated epithelial chemokine expression, and patients displayed a compatible inflammatory stress signature in the serum. Estimated PUFA intake correlated with clinical and biochemical disease activity in a cohort of 160 CD patients, which was similarly demonstrable in an independent metabolomic stool analysis from 199 CD patients. CONCLUSIONS: We provide evidence for the concept of PUFA-induced metabolic gut inflammation which may worsen the course of human CD. Our findings provide a basis for targeted nutritional therapy.
Subject(s)
Crohn Disease , Enteritis , Fatty Acids, Omega-3 , Animals , Crohn Disease/drug therapy , Endoribonucleases , Enteritis/chemically induced , Enteritis/drug therapy , Fatty Acids, Unsaturated , Humans , Inflammation/drug therapy , Mice , Protein Serine-Threonine Kinases , Toll-Like Receptor 2ABSTRACT
Global healthcare systems are challenged by the COVID-19 pandemic. There is a need to optimize allocation of treatment and resources in intensive care, as clinically established risk assessments such as SOFA and APACHE II scores show only limited performance for predicting the survival of severely ill COVID-19 patients. Additional tools are also needed to monitor treatment, including experimental therapies in clinical trials. Comprehensively capturing human physiology, we speculated that proteomics in combination with new data-driven analysis strategies could produce a new generation of prognostic discriminators. We studied two independent cohorts of patients with severe COVID-19 who required intensive care and invasive mechanical ventilation. SOFA score, Charlson comorbidity index, and APACHE II score showed limited performance in predicting the COVID-19 outcome. Instead, the quantification of 321 plasma protein groups at 349 timepoints in 50 critically ill patients receiving invasive mechanical ventilation revealed 14 proteins that showed trajectories different between survivors and non-survivors. A predictor trained on proteomic measurements obtained at the first time point at maximum treatment level (i.e. WHO grade 7), which was weeks before the outcome, achieved accurate classification of survivors (AUROC 0.81). We tested the established predictor on an independent validation cohort (AUROC 1.0). The majority of proteins with high relevance in the prediction model belong to the coagulation system and complement cascade. Our study demonstrates that plasma proteomics can give rise to prognostic predictors substantially outperforming current prognostic markers in intensive care.
ABSTRACT
HDL-mediated cholesterol efflux capacity (CEC) may protect against cardiovascular disease. However, CEC assays are not standardized, hampering their application in large cohorts and comparison between studies. To improve standardization, we systematically investigated technical differences between existing protocols that influence assay performance that have not been previously addressed. CEC was measured in 96-well plates using J774A.1 macrophages labeled with BODIPY-cholesterol and incubated for 4 h with 2% apolipoprotein B-depleted human serum. The time zero method, which calculates CEC using control wells, and the per-well method, which calculates CEC based on the actual content of BODIPY-cholesterol in each well, were compared in 506 samples. We showed that the per-well method had a considerably lower sample rejection rate (4.74% vs. 13.44%) and intra-assay (4.48% vs. 5.28%) and interassay coefficients of variation (two controls: 7.85%, 9.86% vs. 13.58%, 15.29%) compared with the time zero method. Correction for plate-to-plate differences using four controls on each plate also improved assay performance of both methods. In addition, we observed that the lysis reagent used had a significant effect. Compared with cholic acid, lysis with sodium hydroxide results in higher (P = 0.0082) and Triton X-100 in lower (P = 0.0028) CEC values. Furthermore, large cell seeding errors (30% variation) greatly biased CEC for both referencing methods (P < 0.0001) as measured by a resazurin assay. In conclusion, lysis reagents, cell numbers, and assay setup greatly impact the quality and reliability of CEC quantification and should be considered when this method is newly established in a laboratory.
Subject(s)
Cholesterol, HDL/blood , Cholesterol/blood , High-Throughput Screening Assays , Animals , Cell Count , Cell Line , Female , Healthy Volunteers , Humans , Male , MiceABSTRACT
BACKGROUND: Iron deficiency anaemia (IDA) is a major health concern. However, preventive iron supplementation in regions with high burden of infectious diseases resulted in an increase of infection related morbidity and mortality. METHODS: We fed male C57BL/6N mice with either an iron deficient or an iron adequate diet. Next, they received oral iron supplementation or placebo followed by intraperitoneal infection with Salmonella Typhimurium (S.Tm). FINDINGS: We found that mice with IDA had a poorer clinical outcome than mice on an iron adequate diet. Interestingly, iron supplementation of IDA mice resulted in higher bacterial burden in organs and shortened survival. Increased transferrin saturation and non-transferrin bound iron in the circulation together with low expression of ferroportin facilitated the access of the pathogen to iron and promoted bacterial growth. Anaemia, independent of iron supplementation, was correlated with reduced neutrophil counts and cytotoxic T cells. With iron supplementation, anaemia additionally correlated with increased splenic levels of the cytokine IL-10, which is suggestive for a weakened immune control to S.Tm infection. INTERPRETATION: Supplementing iron to anaemic mice worsens the clinical course of bacterial infection. This can be traced back to increased iron delivery to bacteria along with an impaired anti-microbial immune response. Our findings may have important implications for iron supplementation strategies in areas with high endemic burden of infections, putting those individuals, who potentially profit most from iron supplementation for anaemia, at the highest risk for infections. FUNDING: Financial support by the Christian Doppler Laboratory for Iron Metabolism and Anemia Research.
Subject(s)
Anemia, Iron-Deficiency/drug therapy , Bacteremia/complications , Iron/blood , Salmonella Infections/complications , Anemia, Iron-Deficiency/blood , Anemia, Iron-Deficiency/complications , Animals , Bacteremia/blood , Bacteremia/pathology , Bacterial Load , Iron/administration & dosage , Iron/adverse effects , Male , Mice , Mice, Inbred C57BL , Salmonella Infections/blood , Salmonella Infections/pathologyABSTRACT
In this review, we discuss spatiotemporal kinetics and inflammatory signatures of innate immune cells specifically found in response to SARS-CoV-2 compared to influenza virus infection. Importantly, we cover the current understanding on the mechanisms by which SARS-CoV-2 may fail to engage a coordinated type I response and instead may lead to exaggerated inflammation and death. This knowledge is central for the understanding of available data on specialized pro-resolving lipid mediators in severe SARS-CoV-2 infection pointing toward inhibited E-series resolvin synthesis in severe cases. By investigating a publicly available RNA-seq database of bronchoalveolar lavage cells from patients affected by COVID-19, we moreover offer insights into the regulation of key enzymes involved in lipid mediator synthesis, critically complementing the current knowledge about the mediator lipidome in severely affected patients. This review finally discusses different potential approaches to sustain the synthesis of 3-PUFA-derived pro-resolving lipid mediators, including resolvins and lipoxins, which may critically aid in the prevention of acute lung injury and death from COVID-19.
ABSTRACT
Arginase 1 (ARG1) is a cytosolic enzyme that cleaves L-arginine, the substrate of inducible nitric oxide synthase (iNOS), and thereby impairs the control of various intracellular pathogens. Herein, we investigated the role of ARG1 during infection with Salmonella enterica serovar Typhimurium (S.tm). To study the impact of ARG1 on Salmonella infections in vitro, bone marrow-derived macrophages (BMDM) from C57BL/6N wild-type, ARG1-deficient Tie2Cre+/-ARG1fl/fl and NRAMPG169 C57BL/6N mice were infected with S.tm. In wild-type BMDM, ARG1 was induced by S.tm and further upregulated by the addition of interleukin (IL)-4, whereas interferon-γ had an inhibitory effect. Deletion of ARG1 did not result in a reduction in bacterial numbers. In vivo, Arg1 mRNA was upregulated in the spleen, but not in the liver of C57BL/6N mice following intraperitoneal S.tm infection. The genetic deletion of ARG1 (Tie2Cre+/-ARG1fl/fl) or its pharmacological inhibition with CB-1158 neither affected the numbers of S.tm in spleen, liver and blood nor the expression of host response genes such as iNOS, IL-6 or tumour necrosis factor (TNF). Furthermore, ARG1 was dispensable for pathogen control irrespective of the presence or absence of the phagolysosomal natural resistance-associated macrophage protein 1 (NRAMP1). Thus, unlike the detrimental function of ARG1 seen during infections with other intraphagosomal microorganisms, ARG1 did not support bacterial survival in systemic salmonellosis, indicating differential roles of arginine metabolism for host immune response and microbe persistence depending on the type of pathogen.
Subject(s)
Arginase/metabolism , Cytokines/metabolism , Macrophages/metabolism , Macrophages/microbiology , Salmonella Infections, Animal/enzymology , Salmonella typhimurium/physiology , Animals , Bone Marrow Cells/microbiology , Cation Transport Proteins , Integrases/metabolism , Interleukin-4/metabolism , Macrophages/pathology , Mice, Inbred C57BL , Mice, Transgenic , Pyrrolidines/pharmacology , Up-RegulationABSTRACT
Iron is an essential nutrient for mammals as well as for pathogens. Inflammation-driven changes in systemic and cellular iron homeostasis are central for host-mediated antimicrobial strategies. Here, we studied the role of the iron storage protein ferritin H (FTH) for the control of infections with the intracellular pathogen Salmonella enterica serovar Typhimurium by macrophages. Mice lacking FTH in the myeloid lineage (LysM-Cre+/+Fthfl/fl mice) displayed impaired iron storage capacities in the tissue leukocyte compartment, increased levels of labile iron in macrophages, and an accelerated macrophage-mediated iron turnover. While under steady-state conditions, LysM-Cre+/+Fth+/+ and LysM-Cre+/+Fthfl/fl animals showed comparable susceptibility to Salmonella infection, i.v. iron supplementation drastically shortened survival of LysM-Cre+/+Fthfl/fl mice. Mechanistically, these animals displayed increased bacterial burden, which contributed to uncontrolled triggering of NF-κB and inflammasome signaling and development of cytokine storm and death. Importantly, pharmacologic inhibition of the inflammasome and IL-1ß pathways reduced cytokine levels and mortality and partly restored infection control in iron-treated ferritin-deficient mice. These findings uncover incompletely characterized roles of ferritin and cellular iron turnover in myeloid cells in controlling bacterial spread and for modulating NF-κB and inflammasome-mediated cytokine activation, which may be of vital importance in iron-overloaded individuals suffering from severe infections and sepsis.
Subject(s)
Apoferritins , Disease Susceptibility/metabolism , Inflammation , Iron , Macrophages , Salmonella Infections , Salmonella typhimurium/immunology , Animals , Apoferritins/deficiency , Apoferritins/metabolism , Immunity, Innate , Inflammasomes/metabolism , Inflammation/metabolism , Inflammation/microbiology , Interleukin-1beta/immunology , Iron/immunology , Iron/metabolism , Macrophages/immunology , Macrophages/metabolism , Macrophages/microbiology , Mice , Mice, Inbred C57BL , NF-kappa B/metabolism , Salmonella Infections/immunology , Salmonella Infections/metabolism , Signal Transduction/immunologyABSTRACT
COVID-19 is highly variable in its clinical presentation, ranging from asymptomatic infection to severe organ damage and death. We characterized the time-dependent progression of the disease in 139 COVID-19 inpatients by measuring 86 accredited diagnostic parameters, such as blood cell counts and enzyme activities, as well as untargeted plasma proteomes at 687 sampling points. We report an initial spike in a systemic inflammatory response, which is gradually alleviated and followed by a protein signature indicative of tissue repair, metabolic reconstitution, and immunomodulation. We identify prognostic marker signatures for devising risk-adapted treatment strategies and use machine learning to classify therapeutic needs. We show that the machine learning models based on the proteome are transferable to an independent cohort. Our study presents a map linking routinely used clinical diagnostic parameters to plasma proteomes and their dynamics in an infectious disease.
Subject(s)
Biomarkers/analysis , COVID-19/pathology , Disease Progression , Proteome/physiology , Age Factors , Blood Cell Count , Blood Gas Analysis , Enzyme Activation , Humans , Inflammation/pathology , Machine Learning , Prognosis , Proteomics , SARS-CoV-2/immunologyABSTRACT
BACKGROUND: After the 2002/2003 severe acute respiratory syndrome outbreak, 30% of survivors exhibited persisting structural pulmonary abnormalities. The long-term pulmonary sequelae of coronavirus disease 2019 (COVID-19) are yet unknown, and comprehensive clinical follow-up data are lacking. METHODS: In this prospective, multicentre, observational study, we systematically evaluated the cardiopulmonary damage in subjects recovering from COVID-19 at 60 and 100â days after confirmed diagnosis. We conducted a detailed questionnaire, clinical examination, laboratory testing, lung function analysis, echocardiography and thoracic low-dose computed tomography (CT). RESULTS: Data from 145 COVID-19 patients were evaluated, and 41% of all subjects exhibited persistent symptoms 100â days after COVID-19 onset, with dyspnoea being most frequent (36%). Accordingly, patients still displayed an impaired lung function, with a reduced diffusing capacity in 21% of the cohort being the most prominent finding. Cardiac impairment, including a reduced left ventricular function or signs of pulmonary hypertension, was only present in a minority of subjects. CT scans unveiled persisting lung pathologies in 63% of patients, mainly consisting of bilateral ground-glass opacities and/or reticulation in the lower lung lobes, without radiological signs of pulmonary fibrosis. Sequential follow-up evaluations at 60 and 100â days after COVID-19 onset demonstrated a vast improvement of symptoms and CT abnormalities over time. CONCLUSION: A relevant percentage of post-COVID-19 patients presented with persisting symptoms and lung function impairment along with radiological pulmonary abnormalities >100â days after the diagnosis of COVID-19. However, our results indicate a significant improvement in symptoms and cardiopulmonary status over time.
Subject(s)
COVID-19 , Pulmonary Fibrosis , Humans , Lung/diagnostic imaging , Prospective Studies , SARS-CoV-2ABSTRACT
BACKGROUND: Severe coronavirus disease 2019 (COVID-19) is frequently associated with hyperinflammation and hyperferritinemia. The latter is related to increased mortality in COVID-19. Still, it is not clear if iron dysmetabolism is mechanistically linked to COVID-19 pathobiology. METHODS: We herein present data from the ongoing prospective, multicentre, observational CovILD cohort study (ClinicalTrials.gov number, NCT04416100), which systematically follows up patients after COVID-19. 109 participants were evaluated 60 days after onset of first COVID-19 symptoms including clinical examination, chest computed tomography and laboratory testing. RESULTS: We investigated subjects with mild to critical COVID-19, of which the majority received hospital treatment. 60 days after disease onset, 30% of subjects still presented with iron deficiency and 9% had anemia, mostly categorized as anemia of inflammation. Anemic patients had increased levels of inflammation markers such as interleukin-6 and C-reactive protein and survived a more severe course of COVID-19. Hyperferritinemia was still present in 38% of all individuals and was more frequent in subjects with preceding severe or critical COVID-19. Analysis of the mRNA expression of peripheral blood mononuclear cells demonstrated a correlation of increased ferritin and cytokine mRNA expression in these patients. Finally, persisting hyperferritinemia was significantly associated with severe lung pathologies in computed tomography scans and a decreased performance status as compared to patients without hyperferritinemia. DISCUSSION: Alterations of iron homeostasis can persist for at least two months after the onset of COVID-19 and are closely associated with non-resolving lung pathologies and impaired physical performance. Determination of serum iron parameters may thus be a easy to access measure to monitor the resolution of COVID-19. TRIAL REGISTRATION: ClinicalTrials.gov number: NCT04416100.
Subject(s)
Coronavirus Infections/complications , Coronavirus Infections/metabolism , Homeostasis , Iron/metabolism , Lung Diseases/etiology , Lung Diseases/metabolism , Pneumonia, Viral/complications , Pneumonia, Viral/metabolism , Adult , Aged , Anemia/etiology , C-Reactive Protein/analysis , COVID-19 , Cohort Studies , Coronavirus Infections/physiopathology , Female , Ferritins/blood , Follow-Up Studies , Humans , Inflammation/etiology , Inflammation/metabolism , Interleukin-6/blood , Lung Diseases/physiopathology , Male , Middle Aged , Monocytes/metabolism , Pandemics , Pneumonia, Viral/physiopathology , Prospective Studies , Tomography, X-Ray ComputedABSTRACT
There is considerable inter-individual variability in susceptibility to weight gain despite an equally obesogenic environment in large parts of the world. Whereas many studies have focused on identifying the genetic susceptibility to obesity, we performed a GWAS on metabolically healthy thin individuals (lowest 6th percentile of the population-wide BMI spectrum) in a uniquely phenotyped Estonian cohort. We discovered anaplastic lymphoma kinase (ALK) as a candidate thinness gene. In Drosophila, RNAi mediated knockdown of Alk led to decreased triglyceride levels. In mice, genetic deletion of Alk resulted in thin animals with marked resistance to diet- and leptin-mutation-induced obesity. Mechanistically, we found that ALK expression in hypothalamic neurons controls energy expenditure via sympathetic control of adipose tissue lipolysis. Our genetic and mechanistic experiments identify ALK as a thinness gene, which is involved in the resistance to weight gain.
Subject(s)
Anaplastic Lymphoma Kinase/genetics , Thinness/genetics , Adipose Tissue/metabolism , Adult , Animals , Cell Line , Cohort Studies , Drosophila/genetics , Estonia , Female , Humans , Leptin/genetics , Lipolysis/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Obesity/genetics , RNA Interference/physiology , Young AdultABSTRACT
Recombinant erythropoietin (EPO) and iron substitution are a standard of care for treatment of anemias associated with chronic inflammation, including anemia of chronic kidney disease. A black box warning for EPO therapy and concerns about negative side effects related to high-dose iron supplementation as well as the significant proportion of patients becoming EPO resistant over time explains the medical need to define novel strategies to ameliorate anemia of chronic disease (ACD). As hepcidin is central to the iron-restrictive phenotype in ACD, therapeutic approaches targeting hepcidin were recently developed. We herein report the therapeutic effects of a fully human anti-BMP6 antibody (KY1070) either as monotherapy or in combination with Darbepoetin alfa on iron metabolism and anemia resolution in 2 different, well-established, and clinically relevant rodent models of ACD. In addition to counteracting hepcidin-driven iron limitation for erythropoiesis, we found that the combination of KY1070 and recombinant human EPO improved the erythroid response compared with either monotherapy in a qualitative and quantitative manner. Consequently, the combination of KY1070 and Darbepoetin alfa resulted in an EPO-sparing effect. Moreover, we found that suppression of hepcidin via KY1070 modulates ferroportin expression on erythroid precursor cells, thereby lowering potentially toxic-free intracellular iron levels and by accelerating erythroid output as reflected by increased maturation of erythrocyte progenitors. In summary, we conclude that treatment of ACD, as a highly complex disease, becomes more effective by a multifactorial therapeutic approach upon mobilization of endogenous iron deposits and stimulation of erythropoiesis.