Three distinct patterns of mental health response following accidents in mountain sports: a follow-up study of individuals treated at a tertiary trauma center.

The restorative effect of physical activity in alpine environments on mental and physical health is well recognized. However, a risk of accidents and post-accident mental health problems is inherent to every sport. We aimed to characterize mental health in individuals following mountain sport accidents requiring professional medical management. Adult victims of mountain sport accidents treated at the hospital of the Medical University of Innsbruck (Austria) between 2018 and 2020 completed a cross-sectional survey at least 6 months following the admission (median 44 months, n = 307). Symptoms of post-traumatic stress disorder (PTSD, PCL-5), anxiety, depression, and somatization (PHQ), resilience (RS-13), sense of coherence (SOC-9L), post-traumatic growth (PTGI), and quality of life (EUROHIS-QOL), as well as sociodemographic and clinical information, were obtained from an online survey and extracted from electronic health records. Mental health outcome patterns were investigated by semi-supervised medoid clustering and modeled by machine learning. Symptoms of PTSD were observed in 19% of participants. Three comparably sized subsets of participants were identified: a (1) neutral, (2) post-traumatic growth, and (3) post-traumatic stress cluster. The post-traumatic stress cluster was characterized by high prevalence of symptoms of mental disorders, low resilience, low sense of coherence, and low quality of life as well as by younger age, the highest frequency of pre-existing mental disorders, and persisting physical health consequences of the accident. Individuals in this cluster self-reported a need for psychological or psychiatric support following the accident and more cautious behavior during mountain sports since the accident. Reliability of machine learning-based prediction of the cluster assignment based on 40 variables available during acute medical treatment of accident victims was limited. A subset of individuals show symptoms of mental health disorders including symptoms of PTSD when assessed at least 6 months after mountain sport accident. Since early identification of these vulnerable patients remains challenging, psychoeducational measures for all patients and low-threshold access to mental health support are key for a successful interdisciplinary management of victims of mountain sport accidents.

Alterations of blood monocyte subset distribution and surface phenotype are linked to infection severity in COVID-19 inpatients.

Severe coronavirus disease 19 (COVID-19) manifests with systemic immediate proinflammatory innate immune activation and altered iron turnover. Iron homeostasis, differentiation, and function of myeloid leukocytes are interconnected. Therefore, we characterized the cellularity, surface marker expression, and iron transporter phenotype of neutrophils and monocyte subsets in COVID-19 patients within 72 h from hospital admission, and analyzed how these parameters relate to infection severity. Between March and November 2020, blood leukocyte samples from hospitalized COVID-19 patients (n = 48) and healthy individuals (n = 7) were analyzed by flow cytometry enabling comparative analysis of 40 features. Inflammation-driven neutrophil expansion, depletion of CD16+ nonclassical monocytes, and changes in surface expression of neutrophil and monocyte CD64 and CD86 were associated with COVID-19 severity. By unsupervised self-organizing map clustering, four patterns of innate myeloid response were identified and linked to varying levels of systemic inflammation, altered cellular iron trafficking and the severity of disease. These alterations of the myeloid leukocyte compartment during acute COVID-19 may be hallmarks of inefficient viral control and immune hyperactivation and may help at risk prediction and treatment optimization.

Comparative analysis of oral and intravenous iron therapy in rat models of inflammatory anemia and iron deficiency.

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.

The combination of supervised and unsupervised learning based risk stratification and phenotyping in pulmonary arterial hypertension-a long-term retrospective multicenter trial.

BACKGROUND: Accurate risk stratification in pulmonary arterial hypertension (PAH), a devastating cardiopulmonary disease, is essential to guide successful therapy. Machine learning may improve risk management and harness clinical variability in PAH. METHODS: We conducted a long-term retrospective observational study (median follow-up: 67 months) including 183 PAH patients from three Austrian PAH expert centers. Clinical, cardiopulmonary function, laboratory, imaging, and hemodynamic parameters were assessed. Cox proportional hazard Elastic Net and partitioning around medoid clustering were applied to establish a multi-parameter PAH mortality risk signature and investigate PAH phenotypes. RESULTS: Seven parameters identified by Elastic Net modeling, namely age, six-minute walking distance, red blood cell distribution width, cardiac index, pulmonary vascular resistance, N-terminal pro-brain natriuretic peptide and right atrial area, constituted a highly predictive mortality risk signature (training cohort: concordance index = 0.82 [95%CI: 0.75 - 0.89], test cohort: 0.77 [0.66 - 0.88]). The Elastic Net signature demonstrated superior prognostic accuracy as compared with five established risk scores. The signature factors defined two clusters of PAH patients with distinct risk profiles. The high-risk/poor prognosis cluster was characterized by advanced age at diagnosis, poor cardiac output, increased red cell distribution width, higher pulmonary vascular resistance, and a poor six-minute walking test performance. CONCLUSION: Supervised and unsupervised learning algorithms such as Elastic Net regression and medoid clustering are powerful tools for automated mortality risk prediction and clinical phenotyping in PAH.

Distinct smell and taste disorder phenotype of post-acute COVID-19 sequelae.

PURPOSE: Olfactory dysfunction (OD) commonly accompanies coronavirus disease 2019 (COVID-19). We investigated the kinetics of OD resolution following SARS-CoV-2 infection (wild-type and alpha variant) and its impact on quality of life, physical and mental health. METHODS: OD prevalence was assessed in an ambulatory COVID-19 survey (n = 906, ≥ 90 days follow-up) and an observational cohort of ambulatory and hospitalized individuals (n = 108, 360 days follow-up). Co-occurrence of OD with other symptoms and effects on quality of life, physical and mental health were analyzed by multi-dimensional scaling, association rule mining and semi-supervised clustering. RESULTS: Both in the ambulatory COVID-19 survey study (72%) and the observational ambulatory and hospitalized cohort (41%) self-reported OD was frequent during acute COVID-19. Recovery from self-reported OD was slow (survey: median 28 days, observational cohort: 90 days). By clustering of the survey data, we identified a predominantly young, female, comorbidity-free group of convalescents with persistent OD and taste disorders (median recovery: 90 days) but low frequency of post-acute fatigue, respiratory or neurocognitive symptoms. This smell and taste disorder cluster was characterized by a high rating of physical performance, mental health, and quality of life as compared with convalescents affected by prolonged fatigue or neurocognitive complaints. CONCLUSION: Our results underline the heterogeneity of post-acute COVID-19 sequelae calling for tailored management strategies. The persistent smell and taste disorder phenotype is characterized by good clinical, physical, and mental recovery and may pose a minor challenge for public health. STUDY REGISTRATION: ClinicalTrials.gov: NCT04661462 (survey study), NCT04416100 (observational cohort).

Phenotyping of Acute and Persistent Coronavirus Disease 2019 Features in the Outpatient Setting: Exploratory Analysis of an International Cross-sectional Online Survey.

BACKGROUND: Long COVID, defined as the presence of coronavirus disease 2019 (COVID-19) symptoms ≥28 days after clinical onset, is an emerging challenge to healthcare systems. The objective of the current study was to explore recovery phenotypes in nonhospitalized individuals with COVID-19. METHODS: A dual cohort, online survey study was conducted between September 2020 and July 2021 in the neighboring European regions Tyrol (TY; Austria, n = 1157) and South Tyrol (STY; Italy, n = 893). Data were collected on demographics, comorbid conditions, COVID-19 symptoms, and recovery in adult outpatients. Phenotypes of acute COVID-19, postacute sequelae, and risk of protracted recovery were explored using semi-supervised clustering and multiparameter least absolute shrinkage and selection operator (LASSO) modeling. RESULTS: Participants in the study cohorts were predominantly working age (median age [interquartile range], 43 [31-53] years] for TY and 45 [35-55] years] for STY) and female (65.1% in TY and 68.3% in STY). Nearly half (47.6% in TY and 49.3% in STY) reported symptom persistence beyond 28 days. Two acute COVID-19 phenotypes were discerned: the nonspecific infection phenotype and the multiorgan phenotype (MOP). Acute MOP symptoms encompassing multiple neurological, cardiopulmonary, gastrointestinal, and dermatological symptoms were linked to elevated risk of protracted recovery. The major subset of individuals with long COVID (49.3% in TY; 55.6% in STY) displayed no persistent hyposmia or hypogeusia but high counts of postacute MOP symptoms and poor self-reported physical recovery. CONCLUSIONS: The results of our 2-cohort analysis delineated phenotypic diversity of acute and postacute COVID-19 manifestations in home-isolated patients, which must be considered in predicting protracted convalescence and allocating medical resources.

Chest CT of Lung Injury 1 Year after COVID-19 Pneumonia: The CovILD Study.

Background The long-term pulmonary sequelae of COVID-19 is not well known. Purpose To characterize patterns and rates of improvement of chest CT abnormalities 1 year after COVID-19 pneumonia. Materials and Methods This was a secondary analysis of a prospective, multicenter observational cohort study conducted from April 29 to August 12, 2020, to assess pulmonary abnormalities at chest CT approximately 2, 3, and 6 months and 1 year after onset of COVID-19 symptoms. Pulmonary findings were graded for each lung lobe using a qualitative CT severity score (CTSS) ranging from 0 (normal) to 25 (all lobes involved). The association of demographic and clinical factors with CT abnormalities after 1 year was assessed with logistic regression. The rate of change of the CTSS at follow-up CT was investigated by using the Friedmann test. Results Of 142 enrolled participants, 91 underwent a 1-year follow-up CT examination and were included in the analysis (mean age, 59 years ± 13 [SD]; 35 women [38%]). In 49 of 91 (54%) participants, CT abnormalities were observed: 31 of 91 (34%) participants showed subtle subpleural reticulation, ground-glass opacities, or both, and 18 of 91 (20%) participants had extensive ground-glass opacities, reticulations, bronchial dilation, microcystic changes, or a combination thereof. At multivariable analysis, age of more than 60 years (odds ratio [OR], 5.8; 95% CI: 1.7, 24; P = .009), critical COVID-19 severity (OR, 29; 95% CI: 4.8, 280; P < .001), and male sex (OR, 8.9; 95% CI: 2.6, 36; P < .001) were associated with persistent CT abnormalities at 1-year follow-up. Reduction of CTSS was observed in participants at subsequent follow-up CT (P < .001); during the study period, 49% (69 of 142) of participants had complete resolution of CT abnormalities. Thirty-one of 49 (63%) participants with CT abnormalities showed no further improvement after 6 months. Conclusion Long-term CT abnormalities were common 1 year after COVID-19 pneumonia. © RSNA, 2022 Online supplemental material is available for this article. See also the editorial by Leung in this issue.

A fully human anti-BMP6 antibody reduces the need for erythropoietin in rodent models of the anemia of chronic disease.

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.

Factors associated with impaired quality of life three months after being diagnosed with COVID-19.

PURPOSE: To assess patient characteristics associated with health-related quality of life (HR-QoL) and its mental and physical subcategories 3 months after diagnosis with COVID-19. METHODS: In this prospective multicentre cohort study, HR-QoL was assessed in 90 patients using the SF-36 questionnaire (36-item Short Form Health Survey), which consists of 8 health domains that can be divided into a mental and physical health component. Mental health symptoms including anxiety, depression, and post-traumatic stress disorders were evaluated using the Hospital Anxiety and Depression Scale (HADS) and Post-traumatic Stress Disorder Checklist-5 (PCL-5) 3 months after COVID-19. Using descriptive statistics and multivariable regression analysis, we identified factors associated with impaired HR-QoL 3 months after COVID-19 diagnosis. RESULTS: Patients were 55 years of age (IQR, 49-63; 39% women) and were classified as severe (23%), moderate (57%), or mild (20%) according to acute disease severity. HR-QoL was impaired in 28/90 patients (31%). Younger age [per year, adjOR (95%CI) 0.94 (0.88-1.00), p = 0.049], longer hospitalization [per day, adjOR (95%CI) 1.07 (1.01-1.13), p = 0.015], impaired sleep [adjOR (95%CI) 5.54 (1.2-25.61), p = 0.028], and anxiety [adjOR (95%CI) 15.67 (3.03-80.99), p = 0.001) were independently associated with impaired HR-QoL. Twenty-nine percent (n = 26) scored below the normal range on the mental health component of the SF-36 and independent associations emerged for anxiety, depression, and self-reported numbness. Impairments in the physical health component of the SF-36 were reported by 12 (13%) patients and linked to hypogeusia and fatigue. CONCLUSION: Every third patient reported a reduction in HR-QoL 3 months after COVID-19 diagnosis and impairments were more prominent in mental than physical well-being.

Cardiopulmonary recovery after COVID-19: an observational prospective multicentre trial.

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 100 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 100 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 100 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 >100 days after the diagnosis of COVID-19. However, our results indicate a significant improvement in symptoms and cardiopulmonary status over time.

Risk assessment in precapillary pulmonary hypertension: a comparative analysis.

BACKGROUND: Risk stratification is essential to assess mortality risk and guide treatment in patients with precapillary pulmonary hypertension (PH). We herein compared the accuracy of different currently used PH risk stratification tools and evaluated the significance of particular risk parameters. METHODS: We conducted a retrospective longitudinal observational cohort study evaluating seven different risk assessment approaches according to the current PH guidelines. A comprehensive assessment including multi-parametric risk stratification was performed at baseline and 4 yearly follow-up time-points. Multi-step Cox hazard analysis was used to analyse and refine risk prediction. RESULTS: Various available risk models effectively predicted mortality in patients with precapillary pulmonary hypertension. Right-heart catheter parameters were not essential for risk prediction. Contrary, non-invasive follow-up re-evaluations significantly improved the accuracy of risk estimations. A lack of accuracy of various risk models was found in the intermediate- and high-risk classes. For these patients, an additional evaluation step including assessment of age and right atrium area improved risk prediction significantly. DISCUSSION: Currently used abbreviated versions of the ESC/ERS risk assessment tool, as well as the REVEAL 2.0 and REVEAL Lite 2 based risk stratification, lack accuracy to predict mortality in intermediate- and high-risk precapillary pulmonary hypertension patients. An expanded non-invasive evaluation improves mortality risk prediction in these individuals.

The haemochromatosis gene Hfe and Kupffer cells control LDL cholesterol homeostasis and impact on atherosclerosis development.

AIMS: Imbalances of iron metabolism have been linked to the development of atherosclerosis. However, subjects with hereditary haemochromatosis have a lower prevalence of cardiovascular disease. The aim of our study was to understand the underlying mechanisms by combining data from genome-wide association study analyses in humans, CRISPR/Cas9 genome editing, and loss-of-function studies in mice. METHODS AND RESULTS: Our analysis of the Global Lipids Genetics Consortium (GLGC) dataset revealed that single nucleotide polymorphisms (SNPs) in the haemochromatosis gene HFE associate with reduced low-density lipoprotein cholesterol (LDL-C) in human plasma. The LDL-C lowering effect could be phenocopied in dyslipidaemic ApoE-/- mice lacking Hfe, which translated into reduced atherosclerosis burden. Mechanistically, we identified HFE as a negative regulator of LDL receptor expression in hepatocytes. Moreover, we uncovered liver-resident Kupffer cells (KCs) as central players in cholesterol homeostasis as they were found to acquire and transfer LDL-derived cholesterol to hepatocytes in an Abca1-dependent fashion, which is controlled by iron availability. CONCLUSION: Our results disentangle novel regulatory interactions between iron metabolism, KC biology and cholesterol homeostasis which are promising targets for treating dyslipidaemia but also provide a mechanistic explanation for reduced cardiovascular morbidity in subjects with haemochromatosis.

Momelotinib inhibits ACVR1/ALK2, decreases hepcidin production, and ameliorates anemia of chronic disease in rodents.

Patients with myelofibrosis (MF) often develop anemia and frequently become dependent on red blood cell transfusions. Results from a phase 2 study for the treatment of MF with the Janus kinase 1/2 (JAK1/2) inhibitor momelotinib (MMB) demonstrated that MMB treatment ameliorated anemia, which was unexpected for a JAK1/2 inhibitor, because erythropoietin-mediated JAK2 signaling is essential for erythropoiesis. Using a rat model of anemia of chronic disease, we demonstrated that MMB treatment can normalize hemoglobin and red blood cell numbers. We found that this positive effect is driven by direct inhibition of the bone morphogenic protein receptor kinase activin A receptor, type I (ACVR1), and the subsequent reduction of hepatocyte hepcidin production. Of note, ruxolitinib, a JAK1/2 inhibitor approved for the treatment of MF, had no inhibitory activity on this pathway. Further, we demonstrated the effect of MMB is not mediated by direct inhibition of JAK2-mediated ferroportin (FPN1) degradation, because neither MMB treatment nor myeloid-specific deletion of JAK2 affected FPN1 expression. Our data support the hypothesis that the improvement of inflammatory anemia by MMB results from inhibition of ACVR1-mediated hepcidin expression in the liver, which leads to increased mobilization of sequestered iron from cellular stores and subsequent stimulation of erythropoiesis.

Association of mitochondrial iron deficiency and dysfunction with idiopathic restless legs syndrome.

BACKGROUND: Restless legs syndrome is a sensorimotor neurological disorder of the limbs that impairs quality of life and disturbs sleep. However, there has been progress in understanding the disease involving the dopaminergic system as well as iron metabolism. The exact pathophysiological mechanisms of restless legs syndrome remain elusive. We tried to elucidate the underlying mechanisms in iron metabolism in restless legs syndrome subjects on a systemic, cellular, and mitochondrial level. METHODS: We conducted a study prospectively recruiting 168 restless legs syndrome patients and 119 age-matched healthy controls focusing on iron metabolism using human monocytes as surrogates. RESULTS: Evaluation of systemic iron metabolism parameters in the circulation showed no significant difference between patients and controls. We observed a significant reduction in mRNA levels of heme oxygenase 1 and mitochondrial iron genes like mitoferrin 1 and 2 in monocytes isolated from restless legs syndrome patients, indicating mitochondrial iron deficiency. Interestingly, we also observed reduced expression of iron regulatory protein 2 along with impaired activity of mitochondrial aconitase and reduced mitochondrial superoxide formation in restless legs syndrome subjects. Along this line, patients had reduced mitochondrial respiratory capacity that improved in restless legs syndrome subjects under treatment with dopaminergic drugs compared with untreated patients. CONCLUSIONS: Our data suggest that restless legs syndrome is linked to mitochondrial iron deficiency and associated impairment of mitochondrial function. This is partly corrected by treatment with dopaminergic drugs compared with untreated patients, which may be linked to an effect of dopamine on cellular iron homeostasis. © 2018 International Parkinson and Movement Disorder Society.

Salmonella Utilizes Zinc To Subvert Antimicrobial Host Defense of Macrophages via Modulation of NF-κB Signaling.

Zinc sequestration by macrophages is considered a crucial host defense strategy against infection by the intracellular bacterium Salmonella enterica serovar Typhimurium. However, the underlying mechanisms remain elusive. In this study, we found that zinc favors pathogen survival within macrophages. Salmonella-hosting macrophages contained higher free zinc levels than did uninfected macrophages and cells that successfully eliminated bacteria, which was paralleled by the impaired production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in bacterium-harboring cells. A profound, zinc-mediated inhibition of NF-κB p65 transcriptional activity affecting the expression of the ROS- and RNS-forming enzymes phos47 and inducible nitric oxide synthase (iNOS) provided a mechanistic explanation for this phenomenon. Macrophages responded to infection by enhancing the expression of zinc-scavenging metallothioneins 1 and 2, whose genetic deletion caused increased free zinc levels, reduced ROS and RNS production, and increased the survival of Salmonella Our data suggest that Salmonella invasion of macrophages results in a bacterium-driven increase in the intracellular zinc level, which weakens antimicrobial defense and the ability of macrophages to eradicate the pathogen. Thus, limitation of cytoplasmic zinc levels may help to control infection by intracellular bacteria.

Lipocalin 2 drives neutrophilic inflammation in alcoholic liver disease.

BACKGROUND & AIMS: Alcoholic steatohepatitis (ASH) is characterised by neutrophil infiltration that contributes to hepatic injury and disease. Lipocalin-2 (LCN2) was originally identified as siderophore binding peptide in neutrophils, which exerted tissue protective effects in several disease models. Here we investigate the role of LCN2 in the pathogenesis of alcohol-induced liver injury. METHODS: We compared hepatic LCN2 expression in ASH patients, alcoholic cirrhosis patients without evidence of ASH and patients with non-alcoholic fatty liver disease (NAFLD; i.e. simple steatosis). To mechanistically dissect LCN2 function in alcohol-induced liver injury, we subjected wild-type (WT) and Lcn2-deficient (Lcn2(-/-)) mice to the Lieber-DeCarli diet containing 5% ethanol (EtOH) or isocaloric maltose. Adoptive transfer experiments were performed to track neutrophil migration. Furthermore, we tested the effect of antibody-mediated LCN2 neutralisation in an acute model of ethanol-induced hepatic injury. RESULTS: Patients with ASH exhibited increased hepatic LCN2 immunoreactivity compared to patients with alcoholic cirrhosis or simple steatosis, which mainly localised to neutrophils. Similarly, ethanol-fed mice exhibited increased LCN2 expression that mainly localised to leukocytes and especially neutrophils. Lcn2(-/-) mice were protected from alcoholic liver disease (ALD) as demonstrated by reduced neutrophil infiltration, liver injury and hepatic steatosis compared to WT controls. Adoptive transfers revealed that neutrophil-derived LCN2 critically determines hepatic neutrophil immigration and persistence during chronic alcohol exposure. Antibody-mediated neutralisation of LCN2 protected from hepatic injury and neutrophilic infiltration after acute alcohol challenge. CONCLUSIONS: LCN2 drives ethanol-induced neutrophilic inflammation and propagates the development of ALD. Despite a critical role for LCN2 in immunity and infection, pharmacological neutralisation of LCN2 might be of promise in ALD.

In situ proliferation contributes to accumulation of tumor-associated macrophages in spontaneous mammary tumors.

Infiltration of a neoplasm with tumor-associated macrophages (TAMs) is considered an important negative prognostic factor and is functionally associated with tumor vascularization, accelerated growth, and dissemination. However, the ontogeny and differentiation pathways of TAMs are only incompletely characterized. Here, we report that intense local proliferation of fully differentiated macrophages rather than low-pace recruitment of blood-borne precursors drives TAM accumulation in a mouse model of spontaneous mammary carcinogenesis, the MMTVneu strain. TAM differentiation and expansion is regulated by CSF1, whose expression is directly controlled by STAT1 at the gene promoter level. These findings appear to be also relevant for human breast cancer, in which an interrelationship between STAT1, CSF1, and macrophage marker expression was identified. We propose that, akin to various MU subtypes in nonmalignant tissues, local proliferation and CSF1 play a vital role in the homeostasis of TAMs.

Lapatinib and doxorubicin enhance the Stat1-dependent antitumor immune response.

The dual erbB1/2 tyrosine kinase inhibitor lapatinib as well as the anthracycline doxorubicin are both used in the therapy of HER2-positive breast cancer. Using MMTV-neu mice as an animal model for HER2-positive breast cancer, we observed enhanced tumor infiltration by IFN-γ-secreting T cells after treatment with doxorubicin and/or lapatinib. Antibody depletion experiments revealed a contribution of CD8⁺ but not CD4⁺ T cells to the antitumor effect of these drugs. Doxorubicin treatment additionally decreased the content of immunosuppressive tumor-associated macrophages (TAMs) in the tumor bed. In contrast, Stat1-deficient mice were resistant to tumor growth inhibition by lapatinib and/or doxorubicin and exhibited impaired T-cell activation and reduced T-cell infiltration of the tumor in response to drug treatment. Furthermore, Stat1-deficiency resulted in reduced expression of the T-cell chemotactic factors CXCL9, CXCL10, and CXCL11 in the tumor epithelium. The inhibition of TAM infiltration of the tumor by doxorubicin and the immunosuppressive function of TAMs were found to be Stat1 independent. Taken together, the results point to an important contribution toward enhancing T-cell and IFN-γ-based immunity by lapatinib as well as doxorubicin and emphasize the role of Stat1 in building an effective antitumor immune response.

High STAT1 mRNA levels but not its tyrosine phosphorylation are associated with macrophage infiltration and bad prognosis in breast cancer.

BACKGROUND: STAT1 has been attributed a function as tumor suppressor. However, in breast cancer data from microarray analysis indicated a predictive value of high mRNA expression levels of STAT1 and STAT1 target genes belonging to the interferon-related signature for a poor response to therapy. To clarify this issue we have determined STAT1 expression levels and activation by different methods, and investigated their association with tumor infiltration by immune cells. Additionally, we evaluated the interrelationship of these parameters and their significance for predicting disease outcome. METHODS: Expression of STAT1, its target genes SOCS1, IRF1, CXCL9, CXCL10, CXCL11, IFIT1, IFITM1, MX1 and genes characteristic for immune cell infiltration (CD68, CD163, PD-L1, PD-L2, PD-1, CD45, IFN-γ, FOXP3) was determined by RT-PCR in two independent cohorts comprising 132 breast cancer patients. For a subset of patients, protein levels of total as well as serine and tyrosine-phosphorylated STAT1 were ascertained by immunohistochemistry or immunoblotting and protein levels of CXCL10 by ELISA. RESULTS: mRNA expression levels of STAT1 and STAT1 target genes, as well as protein levels of total and serine-phosphorylated STAT1 correlated with each other in neoplastic tissue. However, there was no association between tumor levels of STAT1 mRNA and tyrosine-phosphorylated STAT1 and between CXCL10 serum levels and CXCL10 expression in the tumor. Tumors with increased STAT1 mRNA amounts exhibited elevated expression of genes characteristic for tumor-associated macrophages and immunosuppressive T lymphocytes. Survival analysis revealed an association of high STAT1 mRNA levels and bad prognosis in both cohorts. A similar prognostically relevant correlation with unfavorable outcome was evident for CXCL10, MX1, CD68, CD163, IFN-γ, and PD-L2 expression in at least one collective. By contrast, activation of STAT1 as assessed by the level of STAT1-Y701 phosphorylation was linked to positive outcome. In multivariate Cox regression, the predictive power of STAT1 mRNA expression was lost when including expression of CXCL10, MX1 and CD68 as confounders. CONCLUSIONS: Our study confirms distinct prognostic relevance of STAT1 expression levels and STAT1 tyrosine phosphorylation in breast cancer patients and identifies an association of high STAT1 levels with elevated expression of STAT1 target genes and markers for infiltrating immune cells.