A global effort to dissect the human genetic basis of resistance to SARS-CoV-2 infection.

SARS-CoV-2 infections display tremendous interindividual variability, ranging from asymptomatic infections to life-threatening disease. Inborn errors of, and autoantibodies directed against, type I interferons (IFNs) account for about 20% of critical COVID-19 cases among SARS-CoV-2-infected individuals. By contrast, the genetic and immunological determinants of resistance to infection per se remain unknown. Following the discovery that autosomal recessive deficiency in the DARC chemokine receptor confers resistance to Plasmodium vivax, autosomal recessive deficiencies of chemokine receptor 5 (CCR5) and the enzyme FUT2 were shown to underlie resistance to HIV-1 and noroviruses, respectively. Along the same lines, we propose a strategy for identifying, recruiting, and genetically analyzing individuals who are naturally resistant to SARS-CoV-2 infection.

Untuned antiviral immunity in COVID-19 revealed by temporal type I/III interferon patterns and flu comparison.

A central paradigm of immunity is that interferon (IFN)-mediated antiviral responses precede pro-inflammatory ones, optimizing host protection and minimizing collateral damage1,2. Here, we report that for coronavirus disease 2019 (COVID-19) this paradigm does not apply. By investigating temporal IFN and inflammatory cytokine patterns in 32 moderate-to-severe patients with COVID-19 hospitalized for pneumonia and longitudinally followed for the development of respiratory failure and death, we reveal that IFN-λ and type I IFN production were both diminished and delayed, induced only in a fraction of patients as they became critically ill. On the contrary, pro-inflammatory cytokines such as tumor necrosis factor (TNF), interleukin (IL)-6 and IL-8 were produced before IFNs in all patients and persisted for a prolonged time. This condition was reflected in blood transcriptomes wherein prominent IFN signatures were only seen in critically ill patients who also exhibited augmented inflammation. By comparison, in 16 patients with influenza (flu) hospitalized for pneumonia with similar clinicopathological characteristics to those of COVID-19 and 24 nonhospitalized patients with flu with milder symptoms, IFN-λ and type I IFN were robustly induced earlier, at higher levels and independently of disease severity, whereas pro-inflammatory cytokines were only acutely produced. Notably, higher IFN-λ concentrations in patients with COVID-19 correlated with lower viral load in bronchial aspirates and faster viral clearance and a higher IFN-λ to type I IFN ratio correlated with improved outcome for critically ill patients. Moreover, altered cytokine patterns in patients with COVID-19 correlated with longer hospitalization and higher incidence of critical disease and mortality compared to flu. These data point to an untuned antiviral response in COVID-19, contributing to persistent viral presence, hyperinflammation and respiratory failure.

Type I and type III interferons: From basic biology and genetics to clinical development for COVID-19 and beyond.

Type I and type III interferons (IFNs) constitute a key antiviral defense systems of the body, inducing viral resistance to cells and mediating diverse innate and adaptive immune functions. Defective type I and type III IFN responses have recently emerged as the 'Achilles heel' in COVID-19, with such patients developing severe disease and exhibiting a high risk for critical pneumonia and death. Here, we review the biology of type I and type III IFNs, their similarities and important functional differences, and their roles in SARS-CoV-2 infection. We also appraise the various mechanisms proposed to drive defective IFN responses in COVID-19 with particular emphasis to the ability of SARS-CoV-2 to suppress IFN production and activities, the genetic factors involved and the presence of autoantibodies neutralizing IFNs and accounting for a large proportion of individuals with severe COVID-19. Finally, we discuss the long history of the type I IFN therapeutics for the treatment of viral diseases, cancer and multiple sclerosis, the various efforts to use them in respiratory infections, and the newly emerging type III IFN therapeutics, with emphasis to the more recent studies on COVID-19 and their potential use as broad spectrum antivirals for future epidemics or pandemics.

Interferon-λ Mediates Non-redundant Front-Line Antiviral Protection against Influenza Virus Infection without Compromising Host Fitness.

Lambda interferons (IFNλs) or type III IFNs share homology, expression patterns, signaling cascades, and antiviral functions with type I IFNs. This has complicated the unwinding of their unique non-redundant roles. Through the systematic study of influenza virus infection in mice, we herein show that IFNλs are the first IFNs produced that act at the epithelial barrier to suppress initial viral spread without activating inflammation. If infection progresses, type I IFNs come into play to enhance viral resistance and induce pro-inflammatory responses essential for confronting infection but causing immunopathology. Central to this are neutrophils which respond to both cytokines to upregulate antimicrobial functions but exhibit pro-inflammatory activation only to type I IFNs. Accordingly, Ifnlr1-/- mice display enhanced type I IFN production, neutrophilia, lung injury, and lethality, while therapeutic administration of PEG-IFNλ potently suppresses these effects. IFNλs therefore constitute the front line of antiviral defense in the lung without compromising host fitness.

Notch signaling in adipose tissue macrophages prevents diet-induced inflammation and metabolic dysregulation.

The importance of macrophages in adipose tissue (AT) homeostasis and inflammation is well established. However, the potential cues that regulate their function remain incompletely understood. To bridge this important gap, we sought to characterize novel pathways involved using a mouse model of diet-induced obesity. By performing transcriptomics analysis of AT macrophages (ATMs), we found that late-stage ATMs from high-fat diet mice presented with perturbed Notch signaling accompanied by robust proinflammatory and metabolic changes. To explore the hypothesis that the deregulated Notch pathway contributes to the development of AT inflammation and diet-induced obesity, we employed a genetic approach to abrogate myeloid Notch1 and Notch2 receptors. Our results revealed that the combined loss of Notch1 and Notch2 worsened obesity-related metabolic dysregulation. Body and AT weight gain was higher, blood glucose levels increased and metabolic parameters were substantially worsened in deficient mice fed high-fat diet. Moreover, serum insulin and leptin were elevated as were triglycerides. Molecular analysis of ATMs showed that deletion of Notch receptors escalated inflammation through the induction of an M1-like pro-inflammatory phenotype. Our findings thus support a protective role of myeloid Notch signaling in adipose tissue inflammation and metabolic dysregulation.

Impaired innate antiviral defenses in COVID-19: Causes, consequences and therapeutic opportunities.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently emerged pathogen that has caused coronavirus disease 2019 (COVID-19), the worst pandemic of our times leading to tremendous loss of human life and unprecedented measures of social distancing. COVID-19 symptom manifestations range from asymptomatic disease to severe and lethal outcomes. Lack of previous exposure and immunity to SARS-CoV-2, and high infectivity of the virus have contributed to its broad spread across the globe. In the absence of specific adaptive immunity, innate immune mechanisms are crucial for efficient antiviral defenses and control of the infection. Accumulating evidence now suggests that the remarkable heterogeneity in COVID-19 disease manifestations is due to variable degrees of impairment of innate immune mechanisms. In this review, we summarize recent findings describing both viral and host intrinsic factors that have been linked to defective innate immune responses and account for severe COVID-19. We also discuss emerging therapeutic opportunities for targeting innate immunity for the treatment of COVID-19.

Comprehensive Analysis of 1-Year-Old Female Apolipoprotein E-Deficient Mice Reveals Advanced Atherosclerosis with Vulnerable Plaque Characteristics.

Apolipoprotein E-knockout (Apoe-/-) mice constitute the most widely employed animal model of atherosclerosis. Deletion of Apoe induces profound hypercholesterolemia and promotes the development of atherosclerosis. However, despite its widespread use, the Apoe-/- mouse model remains incompletely characterized, especially at late time points and advanced disease stages. Thus, it is unclear how late atherosclerotic plaques compare to earlier ones in terms of lipid deposition, calcification, macrophage accumulation, smooth muscle cell presence, or plaque necrosis. Additionally, it is unknown how cardiac function and hemodynamic parameters are affected at late disease stages. Here, we used a comprehensive analysis based on histology, fluorescence microscopy, and Doppler ultrasonography to show that in normal chow diet-fed Apoe-/- mice, atherosclerotic lesions at the level of the aortic valve evolve from a more cellular macrophage-rich phenotype at 26 weeks to an acellular, lipid-rich, and more necrotic phenotype at 52 weeks of age, also marked by enhanced lipid deposition and calcification. Coronary artery atherosclerotic lesions are sparse at 26 weeks but ubiquitous and extensive at 52 weeks; yet, left ventricular function was not significantly affected. These findings demonstrate that atherosclerosis in Apoe-/- mice is a highly dynamic process, with atherosclerotic plaques evolving over time. At late disease stages, histopathological characteristics of increased plaque vulnerability predominate in combination with frequent and extensive coronary artery lesions, which nevertheless may not necessarily result in impaired cardiac function.

Respiratory virome profiles reflect antiviral immune responses.

BACKGROUND: From early life, respiratory viruses are implicated in the development, exacerbation and persistence of respiratory conditions such as asthma. Complex dynamics between microbial communities and host immune responses shape immune maturation and homeostasis, influencing health outcomes. We evaluated the hypothesis that the respiratory virome is linked to systemic immune responses, using peripheral blood and nasopharyngeal swab samples from preschool-age children in the PreDicta cohort. METHODS: Peripheral blood mononuclear cells from 51 children (32 asthmatics and 19 healthy controls) participating in the 2-year multinational PreDicta cohort were cultured with bacterial (Bacterial-DNA, LPS) or viral (R848, Poly:IC, RV) stimuli. Supernatants were analysed by Luminex for the presence of 22 relevant cytokines. Virome composition was obtained using untargeted high throughput sequencing of nasopharyngeal samples. The metagenomic data were used for the characterization of virome profiles and the presence of key viral families (Picornaviridae, Anelloviridae, Siphoviridae). These were correlated to cytokine secretion patterns, identified through hierarchical clustering and principal component analysis. RESULTS: High spontaneous cytokine release was associated with increased presence of Prokaryotic virome profiles and reduced presence of Eukaryotic and Anellovirus profiles. Antibacterial responses did not correlate with specific viral families or virome profile; however, low antiviral responders had more Prokaryotic and less Eukaryotic virome profiles. Anelloviruses and Anellovirus-dominated profiles were equally distributed among immune response clusters. The presence of Picornaviridae and Siphoviridae was associated with low interferon-λ responses. Asthma or allergy did not modify these correlations. CONCLUSION: Antiviral cytokine responses at a systemic level reflect the upper airway virome composition. Individuals with low innate interferon responses have higher abundance of Picornaviruses (mostly Rhinoviruses) and bacteriophages. Bacteriophages, particularly Siphoviridae, appear to be sensitive sensors of host antimicrobial capacity, while Anelloviruses are not correlated with TLR-induced immune responses.

Development of an in vitro homeostasis model between airway epithelial cells, bacteria and bacteriophages: a time-lapsed observation of cell viability and inflammatory response.

Bacteriophages represent the most extensive group of viruses within the human virome and have a significant impact on general health and well-being by regulating bacterial population dynamics. Staphylococcus aureus, found in the anterior nostrils, throat and skin, is an opportunistic pathobiont that can cause a wide range of diseases, from chronic inflammation to severe and acute infections. In this study, we developed a human cell-based homeostasis model between a clinically isolated strain of S. aureus 141 and active phages for this strain (PYOSa141) isolated from the commercial Pyophage cocktail (PYO). The cocktail is produced by Eliava BioPreparations Ltd. (Tbilisi, Georgia) and is used as an add-on therapy for bacterial infections, mainly in Georgia. The triptych interaction model was evaluated by time-dependent analysis of cell death and inflammatory response of the nasal and bronchial epithelial cells. Inflammatory mediators (IL-8, CCL5/RANTES, IL-6 and IL-1ß) in the culture supernatants were measured by enzyme-linked immunosorbent assay and cell viability was determined by crystal violet staining. By measuring trans-epithelial electrical resistance, we assessed the epithelial integrity of nasal cells that had differentiated under air-liquid interface conditions. PYOSa141 was found to have a prophylactic effect on airway epithelial cells exposed to S. aureus 141 by effectively down-regulating bacterial-induced inflammation, cell death and epithelial barrier disruption in a time-dependent manner. Overall, the proposed model represents an advance in the way multi-component biological systems can be simulated in vitro.

CD103 integrin identifies a high IL-10-producing FoxP3+ regulatory T-cell population suppressing allergic airway inflammation.

BACKGROUND: Although FoxP3+ regulatory T (Treg) cells constitute a highly heterogeneous population, with different regulatory potential depending on the disease context, distinct subsets or phenotypes remain poorly defined. This hampers the development of immunotherapy for allergic and autoimmune disorders. The present study aimed at characterizing distinct FoxP3+ Treg subpopulations involved in the suppression of Th2-mediated allergic inflammation in the lung. METHODS: We used an established mouse model of allergic airway disease based on ovalbumin sensitization and challenge to analyze FoxP3+ Tregs during the induction and resolution of inflammation, and identify markers that distinguish their most suppressive phenotypes. We also developed a new knock-in mouse model (Foxp3cre Cd103dtr ) enabling the specific ablation of CD103+ FoxP3+ Tregs for functional studies. RESULTS: We found that during resolution of allergic airway inflammation in mice >50% of FoxP3+ Treg cells expressed the integrin CD103 which marks FoxP3+ Treg cells of high IL-10 production, increased expression of immunoregulatory molecules such as KLRG1, ICOS and CD127, and enhanced suppressive capacity for Th2-mediated inflammatory responses. CD103+ FoxP3+ Tregs were essential for keeping allergic inflammation under control as their specific depletion in Foxp3cre Cd103dtr mice lead to severe alveocapillary damage, eosinophilic pneumonia, and markedly reduced lifespan of the animals. Conversely, adoptive transfer of CD103+ FoxP3+ Tregs effectively treated disease, attenuating Th2 responses and allergic inflammation in an IL-10-dependent manner. CONCLUSIONS: Our study identifies a novel regulatory T-cell population, defined by CD103 expression, programmed to prevent exuberant type 2 inflammation and keep homeostasis in the respiratory tract under control. This has important therapeutic implications.

TFEB signaling attenuates NLRP3-driven inflammatory responses in severe asthma.

BACKGROUND: NLRP3-driven inflammatory responses by circulating and lung-resident monocytes are critical drivers of asthma pathogenesis. Autophagy restrains NLRP3-induced monocyte activation in asthma models. Yet, the effects of autophagy and its master regulator, transcription factor EB (TFEB), on monocyte responses in human asthma remain unexplored. Here, we investigated whether activation of autophagy and TFEB signaling suppress inflammatory monocyte responses in asthmatic individuals. METHODS: Peripheral blood CD14+ monocytes from asthmatic patients (n = 83) and healthy controls (n = 46) were stimulated with LPS/ATP to induce NLRP3 activation with or without the autophagy inducer, rapamycin. ASC specks, caspase-1 activation, IL-1ß and IL-18 levels, mitochondrial function, ROS release, and mTORC1 signaling were examined. Autophagy was evaluated by LC3 puncta formation, p62/SQSTM1 degradation and TFEB activation. In a severe asthma (SA) model, we investigated the role of NLRP3 signaling using Nlrp3-/- mice and/or MCC950 administration, and the effects of TFEB activation using myeloid-specific TFEB-overexpressing mice or administration of the TFEB activator, trehalose. RESULTS: We observed increased NLRP3 inflammasome activation, concomitant with impaired autophagy in circulating monocytes that correlated with asthma severity. SA patients also exhibited mitochondrial dysfunction and ROS accumulation. Autophagy failed to inhibit NLRP3-driven monocyte responses, due to defective TFEB activation and excessive mTORC1 signaling. NLRP3 blockade restrained inflammatory cytokine release and linked airway disease. TFEB activation restored impaired autophagy, attenuated NLRP3-driven pulmonary inflammation, and ameliorated SA phenotype. CONCLUSIONS: Our studies uncover a crucial role for TFEB-mediated reprogramming of monocyte inflammatory responses, raising the prospect that this pathway can be therapeutically harnessed for the management of SA.

Prevalence of the BRAF V600E mutation in Greek adults with Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is a rare inflammatory myeloid neoplasia with a broad spectrum of clinical manifestations. The activation of the MAP kinase pathway plays an integral role in its pathogenesis with genetic alterations found in the majority of cases that most frequently involve a somatic mutation of the oncogenic BRAFV600E variant. In this study we investigated the prevalence of the BRAFV600E mutation and its clinical relevance in adult Greek patients with LCH. Among 37 patients studied, the BRAFV600E mutation was identified in 12 out of 31 (38.7%), whereas in six patients (19.3%) the results were in conclusive. The presence of the mutation did not correlate with age at diagnosis, organ involvement, disease extent, response to initial treatment, development of diabetes insipidus and relapse risk. In our series the prevalence of the BRAFV600E mutation is at the lower range of the relative percentage found in children, but in line to that obtained in previous studies of adult patients with LCH that have found an up to 50% prevalence of the BRAFV600E mutation in these patients. Further studies with a larger number of adults are needed to identify the exact prevalence of mutations in the RAS-RAF-MEK-ERK pathway and their role on clinical parameters and disease outcomes.Supplemental data for this article is available online at https://doi.org/10.1080/08880018.2022.2029988 .

Prediction and Ranking of Biomarkers Using multiple UniReD.

Protein-protein interactions (PPIs) are of key importance for understanding how cells and organisms function. Thus, in recent decades, many approaches have been developed for the identification and discovery of such interactions. These approaches addressed the problem of PPI identification either by an experimental point of view or by a computational one. Here, we present an updated version of UniReD, a computational prediction tool which takes advantage of biomedical literature aiming to extract documented, already published protein associations and predict undocumented ones. The usefulness of this computational tool has been previously evaluated by experimentally validating predicted interactions and by benchmarking it against public databases of experimentally validated PPIs. In its updated form, UniReD allows the user to provide a list of proteins of known implication in, e.g., a particular disease, as well as another list of proteins that are potentially associated with the proteins of the first list. UniReD then automatically analyzes both lists and ranks the proteins of the second list by their association with the proteins of the first list, thus serving as a potential biomarker discovery/validation tool.

Differential maturation trajectories of innate antiviral immunity in health and atopy.

BACKGROUND: The maturation of innate immune responses in health and atopy is still incompletely understood. METHODS: We aimed to evaluate age-related trajectories of the TLR3 and TLR7/8 pathways from birth to adulthood and whether these differ between healthy and atopic individuals. Peripheral blood mononuclear cells (PBMCs) were isolated from 39 otherwise healthy, atopic and 39 non-atopic subjects, aged 0-45 years. Selected cytokines involved in antiviral responses were measured by Luminex in culture supernatants of poly(I:C)- and R848-stimulated PBMCs. The non-parametric correlation between age and cytokine expression and differences in developmental trajectories between healthy and atopic subjects were estimated. Patterns of cytokine development were identified with principal component analysis. RESULTS: Normal innate immune maturation entails significant and progressive age-related changes in the production of IL-1ß, TNF-α, MIP-1ß, MCP-3, IP-10, IL-10, IL-12p70, and IFN-γ upon TLR3 and/or TLR7/8 stimulation. Individual cytokines made small contributions to the observed variability; chemokines MCP-3 and IP-10 were key contributors. The development of these pathways deviated in atopic subjects with significant differences observed in the trajectories of IL-1ß, MIP-1ß, and IL-10 syntheses. CONCLUSION: TLR3 and TLR7/8 pathways mature during childhood, while atopy is associated with an abnormal maturation pattern. Suboptimal responses in Th1, inflammatory cytokine, and chemokine production may be implicated in poor antiviral immunity in atopics. Moreover, the deficient maturation of IL-10 synthesis may be implicated in the breaking of tolerance, characterizing the onset of atopic disease.

The TAXINOMISIS Project: A multidisciplinary approach for the development of a new risk stratification model for patients with asymptomatic carotid artery stenosis.

INTRODUCTION: Asymptomatic carotid artery stenosis (ACAS) may cause future stroke and therefore patients with ACAS require best medical treatment. Patients at high risk for stroke may opt for additional revascularization (either surgery or stenting) but the future stroke risk should outweigh the risk for peri/post-operative stroke/death. Current risk stratification for patients with ACAS is largely based on outdated randomized-controlled trials that lack the integration of improved medical therapies and risk factor control. Furthermore, recent circulating and imaging biomarkers for stroke have never been included in a risk stratification model. The TAXINOMISIS Project aims to develop a new risk stratification model for cerebrovascular complications in patients with ACAS and this will be tested through a prospective observational multicentre clinical trial performed in six major European vascular surgery centres. METHODS AND ANALYSIS: The risk stratification model will compromise clinical, circulating, plaque and imaging biomarkers. The prospective multicentre observational study will include 300 patients with 50%-99% ACAS. The primary endpoint is the three-year incidence of cerebrovascular complications. Biomarkers will be retrieved from plasma samples, brain MRI, carotid MRA and duplex ultrasound. The TAXINOMISIS Project will serve as a platform for the development of new computer tools that assess plaque progression based on radiology images and a lab-on-chip with genetic variants that could predict medication response in individual patients. CONCLUSION: Results from the TAXINOMISIS study could potentially improve future risk stratification in patients with ACAS to assist personalized evidence-based treatment decision-making.

Independent association of low IFNλ1 gene expression and type I IFN score/IFNλ1 ratio with obstetric manifestations and triple antiphospholipid antibody positivity in primary antiphospholipid syndrome.

Recent data suggest an important role of type I interferons (IFN) in antiphospholipid syndrome (APS). Here we aimed to evaluate the interplay of type I and type III (or IFNλs) IFNs in APS and potential clinical and serological associations. Our findings suggest that patients with primary APS (PAPS) and systemic lupus erythematosus (SLE)/APS displayed increased type I IFN scores but decreased IFNλ1 gene expression levels compared to healthy individuals, as assessed with real-time qPCR analysis in isolated peripheral blood mononuclear cells (PBMCs). Type I IFN score/IFNλ1 ratio was remarkably higher in patients with PAPS and SLE/APS as well as in SLE patients with or without antiphospholipid antibodies (aPL) vs controls. In conclusion, our results reveal an association between low IFNλ1 expression and obstetric APS. Moreover, the type I IFN score/IFNλ1 ratio seems to be a potential marker of high risk APS given its associations with triple aPL positivity.