Probes for INS

Objectives: Up to 20% and 15% of critically ill influenza and coronavirus disease 2019 (COVID-19) patients are affected by influenza- and COVID-19-associated pulmonary aspergillosis (IAPA and CAPA) respectively. These viral-fungal coinfections are difficult to diagnose and are associated with increased mortality. Mechanistic insights into the development of IAPA and CAPA are a prerequisite for the development of new biomarkers and novel immunomodulatory therapeutic targets. However, data on the pathophysiology are scarce. With this study, we aimed at expanding our knowledge of IAPA and CAPA pathophysiology in an explorative way, resorting to lower respiratory tract samples and focusing on the epithelial and myeloid innate immunity components of the antifungal host response. Methods: We performed nCounter gene expression analyses of 755 genes linked to innate immunity, and determined protein levels of 47 cytokines, chemokines, growth factors, and other inflammatory mediators on bronchoalveolar lavage (BAL) fluid samples from 166 ICU-admitted influenza and COVID-19-patients with or without aspergillosis. Additionally, we performed spatial transcriptomics and RNAscope on in vivo tracheobronchial biopsies from four IAPA and CAPA patients. Results: Several genes encoding proteins with important effector functions in antifungal immunity are downregulated in BAL fluid of IAPA and CAPA patients compared with influenza-only or COVID-19-only patients. Cellular deconvolution of the gene expression data reveals a significantly lower BAL neutrophil fraction in CAPA patients compared to COVID-19-only patients. IAPA and CAPA patients have high BAL fluid levels of pro-inflammatory cytokines, but these are not significantly different from the levels seen in influenza-only and COVID-19-only patients. By integrating the BAL fluid cytokine levels with their respective transcriptional responses, we show that IAPA patients, and to a lesser extent CAPA patients, have an aberrant transcriptional response to pro-inflammatory cytokines as well as type I and type II interferons, which may result in poor cellular effector functions (Fig. 1a). Interferon-gamma signaling is abrogated in both IAPA and CAPA patients when compared with influenza-only and COVID-19-only patients. We observe significantly higher levels of growth factors associated with lung fibrosis in both IAPA and CAPA BAL fluid, which may contribute to the higher mortality seen in these coinfections (Fig. 1b). Using spatial transcriptomics, we show that different epithelial defense mechanisms are at play in IAPA and CAPA (Fig. 2a). Finally, using RNAscope ultrasensitive single-molecule RNA in situ hybridization, we visualize fungal and viral colocalization in CAPA tracheobronchial tissue, proving that virus-induced epithelial barrier disruption paves the way for tissueinvasive aspergillosis (Fig. 2b). Conclusion: Using state-of-the-art techniques in lower respiratory tract samples obtained from a large representative patient cohort, we provide arguments for a three-level breach in antifungal immunity in IAPA and CAPA. A hampered ability to phagocytize and kill fungal spores enables Aspergillus germination and growth, leading to hyphae that are not contained because of restrained extracellular defense mechanisms. These hyphae may easily become tissue-invasive through an epithelium that is weakened by the viral infection, causing detrimental damage to the respiratory system. Functional studies will be necessary to further unravel the pathophysiology of IAPA and CAPA.

Models of heart disease and drug responses are increasingly based on human pluripotent stem cells (hPSCs) since their ability to capture human heart (dys-)function is often better than animal models. Simple monolayer cultures of hPSC-derived cardiomyocytes, however, have shortcomings. Some of these can be overcome using more complex, multi cell-type models in 3D. Here we review modalities that address this, describe efforts to tailor readouts and sensors for monitoring tissue- and cell physiology (exogenously and in situ) and discuss perspectives for implementation in industry and academia.

Hepatic innate immune control of viral infections has largely been attributed to Kupffer cells, the liver macrophages. However, also hepatocytes, the parenchymal cells of the liver, possess potent immunological functions in addition to their known metabolic functions. Owing to their abundance in the liver and known immunological functions, we aimed to investigate the direct anti-viral mechanisms employed by hepatocytes. METHODS: Using lymphocytic choriomeningitis virus (LCMV) as a model of liver infection, we first assessed the role of myeloid cells by depletion prior to infection. We investigated the role of hepatocyte-intrinsic innate immune signaling by infecting mice lacking canonical NF-?B signaling (IKK??Hep) specifically in hepatocytes. In addition, mice lacking hepatocyte-specific interferon-?/? signaling-(IFNAR?Hep), or interferon-?/? signaling in myeloid cells-(IFNAR?Myel) were infected. RESULTS: Here, we demonstrate that LCMV activates NF-?B signaling in hepatocytes. LCMV-triggered NF-?B activation in hepatocytes did not depend on Kupffer cells or TNFR1- but rather on TLR-signaling. LCMV-infected IKK??Hep livers displayed strongly elevated viral titers due to LCMV accumulation within hepatocytes, reduced interferon-stimulated gene (ISG) expression, delayed intrahepatic immune cell influx and delayed intrahepatic LCMV-specific CD8+ T-cell responses. Notably, viral clearance and ISG expression were also reduced in LCMV-infected primary hepatocytes lacking IKK?, demonstrating a hepatocyte-intrinsic effect. Similar to livers of IKK??Hep mice, enhanced hepatocytic LCMV accumulation was observed in livers of IFNAR?Hep, whereas IFNAR?Myel mice were able to control LCMV-infection. Hepatocytic NF-?B signaling was also required for efficient ISG induction in HDV-infected dHepaRG cells and interferon-?/?-mediated inhibition of HBV replication in vitro. CONCLUSIONS: Together, these data show that hepatocyte-intrinsic NF-?B is a vital amplifier of interferon-?/? signaling pivotal for early, strong ISG responses, influx of immune cells and hepatic viral clearance.

Kidney failure is common in patients with Coronavirus Disease-19 (COVID-19) resulting in increased morbidity and mortality. In an international collaboration, 284 kidney biopsies were evaluated to improve understanding of kidney disease in COVID-19. Diagnoses were compared to five years of 63,575 native biopsies prior to the pandemic and 13,955 allograft biopsies to identify diseases increased in patients with COVID-19. Genotyping for APOL1 G1 and G2 alleles was performed in 107 African American and Hispanic patients. Immunohistochemistry for SARS-CoV-2 was utilized to assess direct viral infection in 273 cases along with clinical information at the time of biopsy. The leading indication for native biopsy was acute kidney injury (45.4%), followed by proteinuria with or without concurrent acute kidney injury (42.6%). There were more African American patients (44.6%) than patients of other ethnicities. The most common diagnosis in native biopsies was collapsing glomerulopathy (25.8%) which associated with high-risk APOL1 genotypes in 91.7% of cases. Compared to the five-year biopsy database, the frequency of myoglobin cast nephropathy and proliferative glomerulonephritis with monoclonal IgG deposits was also increased in patients with COVID-19 (3.3% and 1.7%, respectively), while there was a reduced frequency of chronic conditions (including diabetes mellitus, IgA nephropathy, and arterionephrosclerosis) as the primary diagnosis. In transplants, the leading indication was acute kidney injury (86.4%), for which rejection was the predominant diagnosis (61.4%). Direct SARS-CoV-2 viral infection was not identified. Thus, our multi-center large case series identified kidney diseases that disproportionately affect patients with COVID-19, demonstrated a high frequency of APOL1 high-risk genotypes within this group, with no evidence of direct viral infection within the kidney.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes a number of strategies to modulate viral and host mRNA translation. Here, we used ribosome profiling in SARS-CoV-2-infected model cell lines and primary airway cells grown at an air-liquid interface to gain a deeper understanding of the translationally regulated events in response to virus replication. We found that SARS-CoV-2 mRNAs dominate the cellular mRNA pool but are not more efficiently translated than cellular mRNAs. SARS-CoV-2 utilized a highly efficient ribosomal frameshifting strategy despite notable accumulation of ribosomes within the slippery sequence on the frameshifting element. In a highly permissive cell line model, although SARS-CoV-2 infection induced the transcriptional upregulation of numerous chemokine, cytokine, and interferon-stimulated genes, many of these mRNAs were not translated efficiently. The impact of SARS-CoV-2 on host mRNA translation was more subtle in primary cells, with marked transcriptional and translational upregulation of inflammatory and innate immune responses and downregulation of processes involved in ciliated cell function. Together, these data reveal the key role of mRNA translation in SARS-CoV-2 replication and highlight unique mechanisms for therapeutic development. IMPORTANCE SARS-CoV-2 utilizes a number of strategies to modulate host responses to ensure efficient propagation. Here, we used ribosome profiling in SARS-CoV-2-infected cells to gain a deeper understanding of the translationally regulated events in infected cells. We found that although viral mRNAs are abundantly expressed, they are not more efficiently translated than cellular mRNAs. SARS-CoV-2 utilized a highly efficient ribosomal frameshifting strategy and alternative translation initiation sites that help increase the coding potential of its RNAs. In permissive cells, SARS-CoV-2 infection induced the translational repression of numerous innate immune mediators. Though the impact of SARS-CoV-2 on host mRNA translation was more subtle in primary airway cell cultures, we noted marked transcriptional and translational upregulation of inflammatory and innate immune responses and downregulation of processes involved in ciliated cell function. Together, these data provide new insight into how SARS-CoV-2 modulates innate host responses and highlight unique mechanisms for therapeutic intervention.

The Omicron variant has become the most prevalent SARS-CoV-2 variant. Omicron is known to induce milder lesions compared to the original Wuhan strain. Fatal infection of the Wuhan strain into the brain has been well documented in COVID-19 mouse models and human COVID-19 cases, but apparent infections into the brain by Omicron have not been reported in human adult cases or animal models. In this study, we investigated whether Omicron could spread to the brain using K18-hACE2 mice susceptible to SARS-CoV-2 infection.K18-hACE2 mice were intranasally infected with 1 × 105 PFU of the original Wuhan strain and the Omicron variant of SARS-CoV-2. A follow-up was conducted 7 days post infection. All Wuhan-infected mice showed > 20% body weight loss, defined as the lethal condition, whereas two out of five Omicron-infected mice (40%) lost > 20% body weight. Histopathological analysis based on H&E staining revealed inflammatory responses in the brains of these two Omicron-infected mice. Immunostaining analysis of viral nucleocapsid protein revealed severe infection of neuron cells in the brains of these two Omicron-infected mice. Lymphoid depletion and apoptosis were observed in the spleen of Omicron-infected mice with brain infection.Lethal conditions, such as severe body weight loss and encephalopathy, can occur in Omicron-infected K18-hACE2 mice. Our study reports, for the first time, that Omicron can induce brain infection with lymphoid depletion in the mouse COVID-19 model.

The COVID-19 pandemic spurred the rapid development of a range of therapeutic antibody treatments. As part of the US government's COVID-19 therapeutic response, a research team was assembled to support assay and animal model development to assess activity for therapeutics candidates against SARS-CoV-2. Candidate treatments included monoclonal antibodies, antibody cocktails, and products derived from blood donated by convalescent patients. Sixteen candidate antibody products were obtained directly from manufacturers and evaluated for neutralization activity against the WA-01 isolate of SARS-CoV-2. Products were further tested in the Syrian hamster model using prophylactic (-24 h) or therapeutic (+8 h) treatment approaches relative to intranasal SARS-CoV-2 exposure. In vivo assessments included daily clinical scores and body weights. Viral RNA and viable virus titers were quantified in serum and lung tissue with histopathology performed at 3d and 7d post-virus-exposure. Sham-treated, virus-exposed hamsters showed consistent clinical signs with concomitant weight loss and had detectable viral RNA and viable virus in lung tissue. Histopathologically, interstitial pneumonia with consolidation was present. Therapeutic efficacy was identified in treated hamsters by the absence or diminution of clinical scores, body weight loss, viral loads, and improved semiquantitative lung histopathology scores. This work serves as a model for the rapid, systematic in vitro and in vivo assessment of the efficacy of candidate therapeutics at various stages of clinical development. These efforts provided preclinical efficacy data for therapeutic candidates. Furthermore, these studies were invaluable for the phenotypic characterization of SARS CoV-2 disease in hamsters and of utility to the broader scientific community.

Severe COVID-19 lung disease exhibits a high degree of spatial and temporal heterogeneity, with different histological features coexisting within a single individual. It is important to capture the disease complexity to support patient management and treatment strategies. We provide spatially decoded analyses on the immunopathology of diffuse alveolar damage (DAD) patterns and factors that modulate immune and structural changes in fatal COVID-19.We spatially quantified the immune and structural cells in exudative, intermediate, and advanced DAD through multiplex immunohistochemistry in autopsy lung tissue of 18 COVID-19 patients. Cytokine profiling, viral, bacteria, and fungi detection, and transcriptome analyses were performed.Spatial DAD progression was associated with expansion of immune cells, macrophages, CD8+ T cells, fibroblasts, and (lymph)angiogenesis. Viral load correlated positively with exudative DAD and negatively with disease/hospital length. In all cases, enteric bacteria were isolated, and Candida parapsilosis in eight cases. Cytokines correlated mainly with macrophages and CD8+T cells. Pro-coagulation and acute repair were enriched pathways in exudative DAD whereas intermediate/advanced DAD had a molecular profile of elevated humoral and innate immune responses and extracellular matrix production.Unraveling the spatial and molecular immunopathology of COVID-19 cases exposes the responses to SARS-CoV-2-induced exudative DAD and subsequent immune-modulatory and remodeling changes in proliferative/advanced DAD that occur side-by-side together with secondary infections in the lungs. These complex features have important implications for disease management and the development of novel treatments.CNPq, Bill and Melinda Gates Foundation, HC-Convida, FAPESP, Regeneron Pharmaceuticals, and the Swedish Heart & Lung Foundation.

The mammalian placenta, which is responsible for bonding between the mother and the fetus, is one of the first organs to develop. Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection has caused a great threat to public health and affected almost all the organs including the placenta. Owing to limited available data on vertical transmission and pathological changes in the placenta of SARS-CoV-2 positive patients, we aim to review and summarize histopathological and ultrastructural changes in the placental tissue following SARS-CoV-2 infection. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2009 guidelines were used for review writing. Multiple studies have reported significant pathological changes in the placental tissue of SARS-CoV-2 positive mothers. On the other hand, some studies have demonstrated either no or very little involvement of the placental tissue. The most common pathological changes reported are fetal and maternal vascular malformation, villitis of unknown etiology, thrombus formation in the intervillous space and sub-chorionic space, and chorangiosis. Reports on vertical transmission are less in number. The observations of this review present a strong base for the pathological involvement of the placenta in SARS-CoV-2 infected mothers. However, a smaller number of original studies have been done until now, and most of them have small sample sizes and lack matched control groups, which are the big limitations for drawing an effective conclusion at this stage. Antenatal care can be improved by a better understanding of the correlation between maternal SARS-CoV-2 infection and placental pathology in COVID-19.

A T cell-inflamed tumor microenvironment is characterized by the accumulation and local activation of CD8+ T cells and Bat3-lineage dendritic cells, which together are associated with clinical response to anti-programmed cell death protein 1 (anti-PD-1)-based immunotherapy. Preclinical models have demonstrated a crucial role for the chemokine CXCL10 in the recruitment of effector CD8+ T cells into the tumor site, and a chemokine gene signature is also seen in T cell-inflamed tumors from patients. However, the cellular source of CXCL10 in human solid tumors is not known. To identify the cellular source of CXCL10 we analyzed 22 pretreatment biopsy samples of melanoma metastases from patients who subsequently underwent checkpoint blockade immunotherapy. We stained for CD45+ and Sox10+ cells with multiparameter immunofluorescence staining, and RNA in situ hybridization technology was used in concert to identify CXCL10 transcripts. The results were correlated with the expression levels of CXCL10 transcripts from bulk RNA sequencing and the best overall response to immune checkpoint inhibition (anti-PD-1 alone or with anti-CTLA-4) in the same patients. We identified CD45+ cells as the major cellular source for CXCL10 in human melanoma metastases, with additional CXCL10 production seen by Sox10+ cells. Up to 90% of CD45+ cells and up to 69% of Sox10+ cells produced CXCL10 transcripts. The CXCL10 staining result was consistent with the level of CXCL10 expression determined by bulk RNA sequencing. The percentages of CD45+ CXCL10+ cells and Sox10+ CXCL10+ cells independently predicted response (p<0.001). The average number of transcripts per cell correlated with the CD45+ cell infiltrate (R=0.37). Immune cells and melanoma cells produce CXCL10 in human melanoma metastases. Intratumoral CXCL10 is a positive prognostic factor for response to immunotherapy, and the RNAscope technique is achievable using paraffin tissue. Strategies that support effector T cell recruitment via induction of CXCL10 should be considered as a mechanism-based intervention to expand immunotherapy efficacy.

RATIONALE: Patients with idiopathic pulmonary fibrosis (IPF) have worse outcomes following COVID-19. SARSCoV-2 (2019-nCoV) spike protein (S1) harbors an RGD motif in its receptor-binding domain (RBD). Although SARS-CoV-2 is to exploit human Angiotensin Converting Enzyme-2 (ACE2) receptors for cell entry. Single Cell RNA-seq showed that normal lung expresses low levels of ACE2 with very low expression (1.5%) in Alveolar type 2 epithelial cells. It is possible that SARS-CoV-2 needs a cellular co-receptor, which could include integrins, to promote alveolar cell internalization and pneumonitis.METHODS: Solid-phase binding assays were used to investigate S1 binding to ACE2 or αv containing integrins. Pseudovirus entry assays were used to measure the internalization of SARS-CoV-2 into Human embryonic kidney 293T cells expressing different combinations of potential receptors. RNAscope was used to visualize the co-localization of SARS-CoV-2, ACE2, and integrin mRNAs. Immunohistochemistry was used to evaluate the expression of αvβ6 integrins and ACE2 in lung tissue.RESULTS: Binding assays demonstrated that the RGD containing αvβ3 and αvβ6 integrins bound robustly to the SARS-CoV-2 S1 subunit of Spike protein and overexpression of the αvβ6 integrin modestly augments ACE2 mediated SARS-CoV-2 pseudoviral entry into epithelial cells. In COVID-19 damaged lung ACE2 levels are low but the αvβ6 integrin levels are increased in alveolar epithelium whereas both ACE2 and αvβ6 integrin are increased in lung sections from idiopathic pulmonary fibrosis compared with normal lung samples. CONCLUSION: The SARS-CoV-2 S1 subunit can bind αvβ6 integrins augmenting ACE2-dependent internalization of pseudovirus. In IPF patients, ACE2 levels and αvβ6 integrin levels are increased. Increased binding of the SARS-CoV-2 to ACE2 and the αvβ6 integrin within fibrotic lung may explain the increased risk of severe COVID in patients with IPF.