A Mouse-Adapted SARS-CoV-2 Induces Acute Lung Injury and Mortality in Standard Laboratory Mice.

The SARS-CoV-2 pandemic has caused extreme human suffering and economic harm. We generated and characterized a new mouse-adapted SARS-CoV-2 virus that captures multiple aspects of severe COVID-19 disease in standard laboratory mice. This SARS-CoV-2 model exhibits the spectrum of morbidity and mortality of COVID-19 disease as well as aspects of host genetics, age, cellular tropisms, elevated Th1 cytokines, and loss of surfactant expression and pulmonary function linked to pathological features of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). This model can rapidly access existing mouse resources to elucidate the role of host genetics, underlying molecular mechanisms governing SARS-CoV-2 pathogenesis, and the protective or pathogenic immune responses related to disease severity. The model promises to provide a robust platform for studies of ALI and ARDS to evaluate vaccine and antiviral drug performance, including in the most vulnerable populations (i.e., the aged) using standard laboratory mice.

Corrigendum: NLRP12 attenuates colon inflammation by maintaining colonic microbial diversity and promoting protective commensal bacterial growth.

This corrects the article DOI: 10.1038/ni.3690.

NLRP12 attenuates colon inflammation by maintaining colonic microbial diversity and promoting protective commensal bacterial growth.

Inflammatory bowel diseases involve the dynamic interaction of host genetics, the microbiome and inflammatory responses. Here we found lower expression of NLRP12 (which encodes a negative regulator of innate immunity) in human ulcerative colitis, by comparing monozygotic twins and other patient cohorts. In parallel, Nlrp12 deficiency in mice caused increased basal colonic inflammation, which led to a less-diverse microbiome and loss of protective gut commensal strains (of the family Lachnospiraceae) and a greater abundance of colitogenic strains (of the family Erysipelotrichaceae). Dysbiosis and susceptibility to colitis associated with Nlrp12 deficency were reversed equally by treatment with antibodies targeting inflammatory cytokines and by the administration of beneficial commensal Lachnospiraceae isolates. Fecal transplants from mice reared in specific-pathogen-free conditions into germ-free Nlrp12-deficient mice showed that NLRP12 and the microbiome each contributed to immunological signaling that culminated in colon inflammation. These findings reveal a feed-forward loop in which NLRP12 promotes specific commensals that can reverse gut inflammation, while cytokine blockade during NLRP12 deficiency can reverse dysbiosis.

AIM2 in regulatory T cells restrains autoimmune diseases.

The inflammasome initiates innate defence and inflammatory responses by activating caspase-1 and pyroptotic cell death in myeloid cells1,2. It consists of an innate immune receptor/sensor, pro-caspase-1, and a common adaptor molecule, ASC. Consistent with their pro-inflammatory function, caspase-1, ASC and the inflammasome component NLRP3 exacerbate autoimmunity during experimental autoimmune encephalomyelitis by enhancing the secretion of IL-1ß and IL-18 in myeloid cells3-6. Here we show that the DNA-binding inflammasome receptor AIM27-10 has a T cell-intrinsic and inflammasome-independent role in the function of T regulatory (Treg) cells. AIM2 is highly expressed by both human and mouse Treg cells, is induced by TGFß, and its promoter is occupied by transcription factors that are associated with Treg cells such as RUNX1, ETS1, BCL11B and CREB. RNA sequencing, biochemical and metabolic analyses demonstrated that AIM2 attenuates AKT phosphorylation, mTOR and MYC signalling, and glycolysis, but promotes oxidative phosphorylation of lipids in Treg cells. Mechanistically, AIM2 interacts with the RACK1-PP2A phosphatase complex to restrain AKT phosphorylation. Lineage-tracing analysis demonstrates that AIM2 promotes the stability of Treg cells during inflammation. Although AIM2 is generally accepted as an inflammasome effector in myeloid cells, our results demonstrate a T cell-intrinsic role of AIM2 in restraining autoimmunity by reducing AKT-mTOR signalling and altering immune metabolism to enhance the stability of Treg cells.

A mouse-adapted model of SARS-CoV-2 to test COVID-19 countermeasures.

Coronaviruses are prone to transmission to new host species, as recently demonstrated by the spread to humans of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic1. Small animal models that recapitulate SARS-CoV-2 disease are needed urgently for rapid evaluation of medical countermeasures2,3. SARS-CoV-2 cannot infect wild-type laboratory mice owing to inefficient interactions between the viral spike protein and the mouse orthologue of the human receptor, angiotensin-converting enzyme 2 (ACE2)4. Here we used reverse genetics5 to remodel the interaction between SARS-CoV-2 spike protein and mouse ACE2 and designed mouse-adapted SARS-CoV-2 (SARS-CoV-2 MA), a recombinant virus that can use mouse ACE2 for entry into cells. SARS-CoV-2 MA was able to replicate in the upper and lower airways of both young adult and aged BALB/c mice. SARS-CoV-2 MA caused more severe disease in aged mice, and exhibited more clinically relevant phenotypes than those seen in Hfh4-ACE2 transgenic mice, which express human ACE2 under the control of the Hfh4 (also known as Foxj1) promoter. We demonstrate the utility of this model using vaccine-challenge studies in immune-competent mice with native expression of mouse ACE2. Finally, we show that the clinical candidate interferon-λ1a (IFN-λ1a) potently inhibits SARS-CoV-2 replication in primary human airway epithelial cells in vitro-both prophylactic and therapeutic administration of IFN-λ1a diminished SARS-CoV-2 replication in mice. In summary, the mouse-adapted SARS-CoV-2 MA model demonstrates age-related disease pathogenesis and supports the clinical use of pegylated IFN-λ1a as a treatment for human COVID-196.

Host genetic variation guides hepacivirus clearance, chronicity, and liver fibrosis in mice.

BACKGROUND AIMS: Human genetic variation is thought to guide the outcome of HCV infection, but model systems within which to dissect these host genetic mechanisms are limited. Norway rat hepacivirus, closely related to HCV, causes chronic liver infection in rats but causes acute self-limiting hepatitis in typical strains of laboratory mice, which resolves in 2 weeks. The Collaborative Cross (CC) is a robust mouse genetics resource comprised of a panel of recombinant inbred strains, which model the complexity of the human genome and provide a system within which to understand diseases driven by complex allelic variation. APPROACH RESULTS: We infected a panel of CC strains with Norway rat hepacivirus and identified several that failed to clear the virus after 4 weeks. Strains displayed an array of virologic phenotypes ranging from delayed clearance (CC046) to chronicity (CC071, CC080) with viremia for at least 10 months. Body weight loss, hepatocyte infection frequency, viral evolution, T-cell recruitment to the liver, liver inflammation, and the capacity to develop liver fibrosis varied among infected CC strains. CONCLUSIONS: These models recapitulate many aspects of HCV infection in humans and demonstrate that host genetic variation affects a multitude of viruses and host phenotypes. These models can be used to better understand the molecular mechanisms that drive hepacivirus clearance and chronicity, the virus and host interactions that promote chronic disease manifestations like liver fibrosis, therapeutic and vaccine performance, and how these factors are affected by host genetic variation.

Sequence and tissue targeting specificity of ZFP36L2 reveals Elavl2 as a novel target with co-regulation potential.

Zinc finger protein 36 like 2 (ZFP36L2) is an RNA-binding protein that destabilizes transcripts containing adenine-uridine rich elements (AREs). The overlap between ZFP36L2 targets in different tissues is minimal, suggesting that ZFP36L2-targeting is highly tissue specific. We developed a novel Zfp36l2-lacking mouse model (L2-fKO) to identify factors governing this tissue specificity. We found 549 upregulated genes in the L2-fKO spleen by RNA-seq. These upregulated genes were enriched in ARE motifs in the 3'UTRs, which suggests that they are ZFP36L2 targets, however the precise sequence requirement for targeting was not evident from motif analysis alone. We therefore used gel-shift mobility assays on 12 novel putative targets and established that ZFP36L2 requires a 7-mer (UAUUUAU) motif to bind. We observed a statistically significant enrichment of 7-mer ARE motifs in upregulated genes and determined that ZFP36L2 targets are enriched for multiple 7-mer motifs. Elavl2 mRNA, which has three 7-mer (UAUUUAU) motifs, was also upregulated in L2-fKO spleens. Overexpression of ZFP36L2, but not a ZFP36L2(C176S) mutant, reduced Elavl2 mRNA expression, suggesting a direct negative effect. Additionally, a reporter assay demonstrated that the ZFP36L2 effect on Elavl2 decay is dependent on the Elavl2-3'UTR and requires the 7-mer AREs. Our data indicate that Elavl2 mRNA is a novel target of ZFP36L2, specific to the spleen. Likely, ZFP36L2 combined with other RNA binding proteins, such as ELAVL2, governs tissue specificity.

Novel virus-like nanoparticle vaccine effectively protects animal model from SARS-CoV-2 infection.

The key to battling the COVID-19 pandemic and its potential aftermath is to develop a variety of vaccines that are efficacious and safe, elicit lasting immunity, and cover a range of SARS-CoV-2 variants. Recombinant viral receptor-binding domains (RBDs) are safe vaccine candidates but often have limited efficacy due to the lack of virus-like immunogen display pattern. Here we have developed a novel virus-like nanoparticle (VLP) vaccine that displays 120 copies of SARS-CoV-2 RBD on its surface. This VLP-RBD vaccine mimics virus-based vaccines in immunogen display, which boosts its efficacy, while maintaining the safety of protein-based subunit vaccines. Compared to the RBD vaccine, the VLP-RBD vaccine induced five times more neutralizing antibodies in mice that efficiently blocked SARS-CoV-2 from attaching to its host receptor and potently neutralized the cell entry of variant SARS-CoV-2 strains, SARS-CoV-1, and SARS-CoV-1-related bat coronavirus. These neutralizing immune responses induced by the VLP-RBD vaccine did not wane during the two-month study period. Furthermore, the VLP-RBD vaccine effectively protected mice from SARS-CoV-2 challenge, dramatically reducing the development of clinical signs and pathological changes in immunized mice. The VLP-RBD vaccine provides one potentially effective solution to controlling the spread of SARS-CoV-2.

Targeted inhibition of gut bacterial ß-glucuronidase activity enhances anticancer drug efficacy.

Irinotecan treats a range of solid tumors, but its effectiveness is severely limited by gastrointestinal (GI) tract toxicity caused by gut bacterial ß-glucuronidase (GUS) enzymes. Targeted bacterial GUS inhibitors have been shown to partially alleviate irinotecan-induced GI tract damage and resultant diarrhea in mice. Here, we unravel the mechanistic basis for GI protection by gut microbial GUS inhibitors using in vivo models. We use in vitro, in fimo, and in vivo models to determine whether GUS inhibition alters the anticancer efficacy of irinotecan. We demonstrate that a single dose of irinotecan increases GI bacterial GUS activity in 1 d and reduces intestinal epithelial cell proliferation in 5 d, both blocked by a single dose of a GUS inhibitor. In a tumor xenograft model, GUS inhibition prevents intestinal toxicity and maintains the antitumor efficacy of irinotecan. Remarkably, GUS inhibitor also effectively blocks the striking irinotecan-induced bloom of Enterobacteriaceae in immune-deficient mice. In a genetically engineered mouse model of cancer, GUS inhibition alleviates gut damage, improves survival, and does not alter gut microbial composition; however, by allowing dose intensification, it dramatically improves irinotecan's effectiveness, reducing tumors to a fraction of that achieved by irinotecan alone, while simultaneously promoting epithelial regeneration. These results indicate that targeted gut microbial enzyme inhibitors can improve cancer chemotherapeutic outcomes by protecting the gut epithelium from microbial dysbiosis and proliferative crypt damage.

Update on the Features and Measurements of Experimental Acute Lung Injury in Animals: An Official American Thoracic Society Workshop Report.

Advancements in methods, technology, and our understanding of the pathobiology of lung injury have created the need to update the definition of experimental acute lung injury (ALI). We queried 50 participants with expertise in ALI and acute respiratory distress syndrome using a Delphi method composed of a series of electronic surveys and a virtual workshop. We propose that ALI presents as a "multidimensional entity" characterized by four "domains" that reflect the key pathophysiologic features and underlying biology of human acute respiratory distress syndrome. These domains are 1) histological evidence of tissue injury, 2) alteration of the alveolar-capillary barrier, 3) presence of an inflammatory response, and 4) physiologic dysfunction. For each domain, we present "relevant measurements," defined as those proposed by at least 30% of respondents. We propose that experimental ALI encompasses a continuum of models ranging from those focusing on gaining specific mechanistic insights to those primarily concerned with preclinical testing of novel therapeutics or interventions. We suggest that mechanistic studies may justifiably focus on a single domain of lung injury, but models must document alterations of at least three of the four domains to qualify as "experimental ALI." Finally, we propose that a time criterion defining "acute" in ALI remains relevant, but the actual time may vary based on the specific model and the aspect of injury being modeled. The continuum concept of ALI increases the flexibility and applicability of the definition to multiple models while increasing the likelihood of translating preclinical findings to critically ill patients.

IL-1α Is Essential for Oviduct Pathology during Genital Chlamydial Infection in Mice.

Chlamydia trachomatis infection of the female genital tract can lead to irreversible fallopian tube scarring. In the mouse model of genital infection using Chlamydia muridarum, IL-1R signaling plays a critical role in oviduct tissue damage. In this study, we investigated the pathologic role of IL-1α, one of the two proinflammatory cytokines that bind to IL-1R. Il1a-/- mice infected with C. muridarum cleared infection at their cervix at the same rate as wild-type (WT) mice, but were significantly protected from end point oviduct damage and fibrosis. The contribution of IL-1α to oviduct pathology was more dramatic than observed in mice deficient for IL-1ß. Although chlamydial burden was similar in WT and Il1a-/- oviduct during peak days of infection, levels of IL-1ß, IL-6, CSF3, and CXCL2 were reduced in Il1a-/- oviduct lysates. During infection, Il1a-/- oviducts and uterine horns exhibited reduced neutrophil infiltration, and this reduction persisted after the infection resolved. The absence of IL-1α did not compromise CD4 T cell recruitment or function during primary or secondary chlamydial infection. IL-1α is expressed predominantly by luminal cells of the genital tract in response to infection, and low levels of expression persisted after the infection cleared. Ab-mediated depletion of IL-1α in WT mice prevented infection-induced oviduct damage, further supporting a key role for IL-1α in oviduct pathology.

Preclinical coronavirus studies and pathology: Challenges of the high-containment laboratory.

The COVID-19 pandemic has highlighted the critical role that animal models play in elucidating the pathogenesis of emerging diseases and rapidly analyzing potential medical countermeasures. Relevant pathologic outcomes are paramount in evaluating preclinical models and therapeutic outcomes and require careful advance planning. While there are numerous guidelines for attaining high-quality pathology specimens in routine animal studies, preclinical studies using coronaviruses are often conducted under biosafety level-3 (BSL3) conditions, which pose unique challenges and technical limitations. In such settings, rather than foregoing pathologic outcomes because of the inherent constraints of high-containment laboratory protocols, modifications can be made to conventional best practices of specimen collection. Particularly for those unfamiliar with working in a high-containment laboratory, the authors describe the logistics of conducting such work, focusing on animal experiments in BSL3 conditions. To promote scientific rigor and reproducibility and maximize the value of animal use, the authors provide specific points to be considered before, during, and following a high-containment animal study. The authors provide procedural modifications for attaining good quality pathologic assessment of the mouse lung, central nervous system, and blood specimens under high-containment conditions while being conscientious to maximize animal use for other concurrent assays.

One-pot synthesis of carboxymethyl-dextran coated iron oxide nanoparticles (CION) for preclinical fMRI and MRA applications.

Superparamagnetic iron-oxide nanoparticles are robust contrast agents for magnetic resonance imaging (MRI) used for sensitive structural and functional mapping of the cerebral blood volume (CBV) when administered intravenously. To date, many CBV-MRI studies are conducted with Feraheme, manufactured for the clinical treatment of iron-deficiency. Unfortunately, Feraheme is currently not available outside the United States due to commercial and regulatory constraints, making CBV-MRI methods either inaccessible or very costly to achieve. To address this barrier, we developed a simple, one-pot recipe to synthesize Carboxymethyl-dextran coated Iron Oxide Nanoparticles, namely, "CION", suitable for preclinical CBV-MRI applications. Here we disseminate a step-by-step instruction of our one-pot synthesis protocol, which allows CION to be produced in laboratories with minimal cost. We also characterized different CION-conjugations by manipulating polymer to metal stoichiometric ratio in terms of their size, surface chemistry, and chemical composition, and shifts in MR relaxivity and pharmacokinetics. We performed several proof-of-concept experiments in vivo, demonstrating the utility of CION for functional and structural MRI applications, including hypercapnic CO2 challenge, visual stimulation, targeted optogenetic stimulation, and microangiography. We also present evidence that CION can serve as a cross-modality research platform by showing concurrent in vivo optical and MRI measurement of CBV using fluorescent-labeled CION. The simplicity and cost-effectiveness of our one-pot synthesis method should allow researchers to reproduce CION and tailor the relaxivity and pharmacokinetics according to their imaging needs. It is our hope that this work makes CBV-MRI more openly available and affordable for a variety of research applications.

A conditional mouse expressing an activating mutation in NRF2 displays hyperplasia of the upper gastrointestinal tract and decreased white adipose tissue.

Activation of the nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or NRF2) transcription factor is a critical and evolutionarily conserved cellular response to oxidative stress, metabolic stress, and xenobiotic insult. Deficiency of NRF2 results in hypersensitivity to a variety of stressors, whereas its aberrant activation contributes to several cancer types, most commonly squamous cell carcinomas of the esophagus, oral cavity, bladder, and lung. Between 10% and 35% of patients with squamous cell carcinomas display hyperactive NRF2 signaling, harboring activating mutations and copy number amplifications of the NFE2L2 oncogene or inactivating mutations or deletions of KEAP1 or CUL3, the proteins of which co-complex to ubiquitylate and degrade NRF2 protein. To better understand the role of NRF2 in tumorigenesis and more broadly in development, we engineered the endogenous Nfe2l2 genomic locus to create a conditional mutant LSL-Nrf2E79Q mouse model. The E79Q mutation, one of the most commonly observed NRF2-activating mutations in human squamous cancers, codes for a mutant protein that does not undergo KEAP1/CUL3-dependent degradation, resulting in its constitutive activity. Expression of NRF2 E79Q protein in keratin 14 (KRT14)-positive murine tissues resulted in hyperplasia of squamous cell tissues of the tongue, forestomach, and esophagus, a stunted body axis, decreased weight, and decreased visceral adipose depots. RNA-seq profiling and follow-up validation studies of cultured NRF2E79Q murine esophageal epithelial cells revealed known and novel NRF2-regulated transcriptional programs, including genes associated with squamous cell carcinoma (e.g. Myc), lipid and cellular metabolism (Hk2, Ppard), and growth factors (Areg, Bmp6, Vegfa). These data suggest that in addition to decreasing adipogenesis, KRT14-restricted NRF2 activation drives hyperplasia of the esophagus, forestomach, and tongue, but not formation of squamous cell carcinoma. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Biodistribution, cardiac and neurobehavioral assessments, and neurotransmitter quantification in juvenile rats following oral administration of aluminum oxide nanoparticles.

Little is known about the uptake, biodistribution, and biological responses of nanoparticles (NPs) and their toxicity in developing animals. Here, male and female juvenile Sprague-Dawley rats received four consecutive daily doses of 10 mg/kg Al2 O3 NP (diameter: 24 nm [transmission electron microscope], hydrodynamic diameter: 148 nm) or vehicle control (water) by gavage between postnatal days (PNDs) 17-20. Basic neurobehavioral and cardiac assessments were performed on PND 20. Animals were sacrificed on PND 21, and selected tissues were collected, weighed, and processed for histopathology or neurotransmitter analysis. The biodistribution of Al2 O3 NP in tissue sections of the intestine, liver, spleen, kidney, and lymph nodes were evaluated using enhanced dark-field microscopy (EDM) and hyperspectral imaging (HSI). Liver-to-body weight ratio was significantly increased for male pups administered Al2 O3 NP compared with control. HSI suggested that Al2 O3 NP was more abundant in the duodenum and ileum tissue of the female pups compared with the male pups, whereas the abundance of NP was similar for males and females in the other tissues. The abundance of NP was higher in the liver compared with spleen, lymph nodes, and kidney. Homovanillic acid and norepinephrine concentrations in brain were significantly decreased following Al2 O3 NP administration in female and male pups, whereas 5-hydroxyindoleacetic acid was significantly increased in male pups. EDM/HSI indicates intestinal uptake of Al2 O3 NP following oral administration. Al2 O3 NP altered neurotransmitter/metabolite concentrations in juvenile rats' brain tissues. Together, these data suggest that orally administered Al2 O3 NP interferes with the brain biochemistry in both female and male pups.

In vivo assessment of pulmonary fibrosis and edema in rodents using the backscatter coefficient and envelope statistics.

Quantitative ultrasound methods based on the backscatter coefficient (BSC) and envelope statistics have been used to quantify disease in a wide variety of tissues, such as prostate, lymph nodes, breast, and thyroid. However, to date, these methods have not been investigated in the lung. In this study, lung properties were quantified by BSC and envelope statistical parameters in normal, fibrotic, and edematous rat lungs in vivo. The average and standard deviation of each parameter were calculated for each lung as well as the evolution of each parameter with acoustic propagation time within the lung. The transport mean free path and backscattered frequency shift, two parameters that have been successfully used to assess pulmonary fibrosis and edema in prior work, were evaluated in combination with the BSC and envelope statistical parameters. Multiple BSC and envelope statistical parameters were found to provide contrast between control and diseased lungs. BSC and envelope statistical parameters were also significantly correlated with fibrosis severity using the modified Ashcroft fibrosis score as the histological gold standard. These results demonstrate the potential for BSC and envelope statistical parameters to improve the diagnosis of pulmonary fibrosis and edema as well as monitor pulmonary fibrosis.

Ursodeoxycholic Acid (UDCA) Mitigates the Host Inflammatory Response during Clostridioides difficile Infection by Altering Gut Bile Acids.

Clostridioides difficile infection (CDI) is associated with increasing morbidity and mortality posing an urgent threat to public health. Recurrence of CDI after successful treatment with antibiotics is high, thus necessitating discovery of novel therapeutics against this enteric pathogen. Administration of the secondary bile acid ursodeoxycholic acid (UDCA; ursodiol) inhibits the life cycles of various strains of C. difficilein vitro, suggesting that the FDA-approved formulation of UDCA, known as ursodiol, may be able to restore colonization resistance against C. difficilein vivo However, the mechanism(s) by which ursodiol is able to restore colonization resistance against C. difficile remains unknown. Here, we confirmed that ursodiol inhibits C. difficile R20291 spore germination and outgrowth, growth, and toxin activity in a dose-dependent manner in vitro In a murine model of CDI, exogenous administration of ursodiol resulted in significant alterations in the bile acid metabolome with little to no changes in gut microbial community structure. Ursodiol pretreatment resulted in attenuation of CDI pathogenesis early in the course of disease, which coincided with alterations in the cecal and colonic inflammatory transcriptome, bile acid-activated receptors nuclear farnesoid X receptor (FXR) and transmembrane G-protein-coupled membrane receptor 5 (TGR5), which are able to modulate the innate immune response through signaling pathways such as NF-κB. Although ursodiol pretreatment did not result in a consistent decrease in the C. difficile life cycle in vivo, it was able to attenuate an overly robust inflammatory response that is detrimental to the host during CDI. Ursodiol remains a viable nonantibiotic treatment and/or prevention strategy against CDI. Likewise, modulation of the host innate immune response via bile acid-activated receptors FXR and TGR5 represents a new potential treatment strategy for patients with CDI.