NET formation is a default epigenetic program controlled by PAD4 in apoptotic neutrophils.

Neutrophil extracellular traps (NETs) not only counteract bacterial and fungal pathogens but can also promote thrombosis, autoimmunity, and sterile inflammation. The presence of citrullinated histones, generated by the peptidylarginine deiminase 4 (PAD4), is synonymous with NETosis and is considered independent of apoptosis. Mitochondrial- and death receptor-mediated apoptosis promote gasdermin E (GSDME)-dependent calcium mobilization and membrane permeabilization leading to histone H3 citrullination (H3Cit), nuclear DNA extrusion, and cytoplast formation. H3Cit is concentrated at the promoter in bone marrow neutrophils and redistributes in a coordinated process from promoter to intergenic and intronic regions during apoptosis. Loss of GSDME prevents nuclear and plasma membrane disruption of apoptotic neutrophils but prolongs early apoptosis-induced cellular changes to the chromatin and cytoplasmic granules. Apoptotic signaling engages PAD4 in neutrophils, establishing a cellular state that is primed for NETosis, but that occurs only upon membrane disruption by GSDME, thereby redefining the end of life for neutrophils.

The lung employs an intrinsic surfactant-mediated inflammatory response for viral defense.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) causes an acute respiratory distress syndrome (ARDS) that resembles surfactant deficient RDS. Using a novel multi-cell type, human induced pluripotent stem cell (hiPSC)-derived lung organoid (LO) system, validated against primary lung cells, we found that inflammatory cytokine/chemokine production and interferon (IFN) responses are dynamically regulated autonomously within the lung following SARS-CoV-2 infection, an intrinsic defense mechanism mediated by surfactant proteins (SP). Single cell RNA sequencing revealed broad infectability of most lung cell types through canonical (ACE2) and non-canonical (endocytotic) viral entry routes. SARS-CoV-2 triggers rapid apoptosis, impairing viral dissemination. In the absence of surfactant protein B (SP-B), resistance to infection was impaired and cytokine/chemokine production and IFN responses were modulated. Exogenous surfactant, recombinant SP-B, or genomic correction of the SP-B deletion restored resistance to SARS-CoV-2 and improved viability.

NLRP3 activation in neutrophils induces lethal autoinflammation, liver inflammation, and fibrosis.

Sterile inflammation is a central element in liver diseases. The immune response following injurious stimuli involves hepatic infiltration of neutrophils and monocytes. Neutrophils are major effectors of liver inflammation, rapidly recruited to sites of inflammation, and can augment the recruitment of other leukocytes. The NLRP3 inflammasome has been increasingly implicated in severe liver inflammation, fibrosis, and cell death. In this study, the role of NLRP3 activation in neutrophils during liver inflammation and fibrosis was investigated. Mouse models with neutrophil-specific expression of mutant NLRP3 were developed. Mutant mice develop severe liver inflammation and lethal autoinflammation phenocopying mice with a systemic expression of mutant NLRP3. NLRP3 activation in neutrophils leads to a pro-inflammatory cytokine and chemokine profile in the liver, infiltration by neutrophils and macrophages, and an increase in cell death. Furthermore, mutant mice develop liver fibrosis associated with increased expression of pro-fibrogenic genes. Taken together, the present work demonstrates how neutrophils, driven by the NLRP3 inflammasome, coordinate other inflammatory myeloid cells in the liver, and propagate the inflammatory response in the context of inflammation-driven fibrosis.

Conformational flexibility in neutralization of SARS-CoV-2 by naturally elicited anti-SARS-CoV-2 antibodies.

As new variants of SARS-CoV-2 continue to emerge, it is important to assess the cross-neutralizing capabilities of antibodies naturally elicited during wild type SARS-CoV-2 infection. In the present study, we evaluate the activity of nine anti-SARS-CoV-2 monoclonal antibodies (mAbs), previously isolated from convalescent donors infected with the Wuhan-Hu-1 strain, against the SARS-CoV-2 variants of concern (VOC) Alpha, Beta, Gamma, Delta and Omicron. By testing an array of mutated spike receptor binding domain (RBD) proteins, cell-expressed spike proteins from VOCs, and neutralization of SARS-CoV-2 VOCs as pseudoviruses, or as the authentic viruses in culture, we show that mAbs directed against the ACE2 binding site (ACE2bs) are more sensitive to viral evolution compared to anti-RBD non-ACE2bs mAbs, two of which retain their potency against all VOCs tested. At the second part of our study, we reveal the neutralization mechanisms at high molecular resolution of two anti-SARS-CoV-2 neutralizing mAbs by structural characterization. We solve the structures of the Delta-neutralizing ACE2bs mAb TAU-2303 with the SARS-CoV-2 spike trimer and RBD at 4.5 Å and 2.42 Å resolutions, respectively, revealing a similar mode of binding to that between the RBD and ACE2. Furthermore, we provide five additional structures (at resolutions of 4.7 Å, 7.3 Å, 6.4 Å, 3.3 Å, and 6.1 Å) of a second antibody, TAU-2212, complexed with the SARS-CoV-2 spike trimer. TAU-2212 binds an exclusively quaternary epitope, and exhibits a unique, flexible mode of neutralization that involves transitioning between five different conformations, with both arms of the antibody recruited for cross linking intra- and inter-spike RBD subunits. Our study provides additional mechanistic understanding about how antibodies neutralize SARS-CoV-2 and its emerging variants and provides insights on the likelihood of reinfections.

Non-apoptotic Cell Death Control of Neutrophil Extracellular Trap Formation.

Neutrophil extracellular traps (NETs) are networks of chromatin and microbicidal proteins released by neutrophils in response to infection and tissue damage. Although classically viewed as a discrete biochemical and cellular process in neutrophils, the effector pathways integrating diverse upstream activating signals to control the formation of NETs (NETosis) are poorly defined. Cell death is one such common unifying endpoint of neutrophils, with several bona fide non-apoptotic cell death agonists now described to initiate NETosis. Integrating these new genetic findings into our existing knowledge of NETosis will likely reveal varied cellular and biochemical processes controlling NET release and specific anti-microbial and inflammatory effector functions of NETs triggered by specific non-apoptotic cell death. To facilitate investigation of regulated cell death pathways in NETosis, we offer a detailed protocol for neutrophil purification from mouse bone marrow and human blood, analysis of NETs by flow cytometry, and validation by immunogold electron microscopy. Future studies may better define cell death-specific forms of NETosis and their influence on inflammation and autoimmunity.

Discovery and functional interrogation of SARS-CoV-2 protein-RNA interactions.

The COVID-19 pandemic is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The betacoronvirus has a positive sense RNA genome which encodes for several RNA binding proteins. Here, we use enhanced crosslinking and immunoprecipitation to investigate SARS-CoV-2 protein interactions with viral and host RNAs in authentic virus-infected cells. SARS-CoV-2 proteins, NSP8, NSP12, and nucleocapsid display distinct preferences to specific regions in the RNA viral genome, providing evidence for their shared and separate roles in replication, transcription, and viral packaging. SARS-CoV-2 proteins expressed in human lung epithelial cells bind to 4773 unique host coding RNAs. Nine SARS-CoV-2 proteins upregulate target gene expression, including NSP12 and ORF9c, whose RNA substrates are associated with pathways in protein N-linked glycosylation ER processing and mitochondrial processes. Furthermore, siRNA knockdown of host genes targeted by viral proteins in human lung organoid cells identify potential antiviral host targets across different SARS-CoV-2 variants. Conversely, NSP9 inhibits host gene expression by blocking mRNA export and dampens cytokine productions, including interleukin-1α/ß. Our viral protein-RNA interactome provides a catalog of potential therapeutic targets and offers insight into the etiology of COVID-19 as a safeguard against future pandemics.

Walking down the memory lane with SARS-CoV-2 B cells.

The B-cell response to COVID-19 vaccines in convalescent individuals.

Age-dependent regulation of SARS-CoV-2 cell entry genes and cell death programs correlates with COVID-19 severity.

Novel coronavirus disease 2019 (COVID-19) severity is highly variable, with pediatric patients typically experiencing less severe infection than adults and especially the elderly. The basis for this difference is unclear. We find that mRNA and protein expression of angiotensin-converting enzyme 2 (ACE2), the cell entry receptor for the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes COVID-19, increases with advancing age in distal lung epithelial cells. However, in humans, ACE2 expression exhibits high levels of intra- and interindividual heterogeneity. Further, cells infected with SARS-CoV-2 experience endoplasmic reticulum stress, triggering an unfolded protein response and caspase-mediated apoptosis, a natural host defense system that halts virion production. Apoptosis of infected cells can be selectively induced by treatment with apoptosis-modulating BH3 mimetic drugs. Notably, epithelial cells within young lungs and airways are more primed to undergo apoptosis than those in adults, which may naturally hinder virion production and support milder COVID-19 severity.

Immune response to intravenous immunoglobulin in patients with Kawasaki disease and MIS-C.

BACKGROUNDMultisystem inflammatory syndrome in children (MIS-C) is a rare but potentially severe illness that follows exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Kawasaki disease (KD) shares several clinical features with MIS-C, which prompted the use of intravenous immunoglobulin (IVIG), a mainstay therapy for KD. Both diseases share a robust activation of the innate immune system, including the IL-1 signaling pathway, and IL-1 blockade has been used for the treatment of both MIS-C and KD. The mechanism of action of IVIG in these 2 diseases and the cellular source of IL-1ß have not been defined.METHODSThe effects of IVIG on peripheral blood leukocyte populations from patients with MIS-C and KD were examined using flow cytometry and mass cytometry (CyTOF) and live-cell imaging.RESULTSCirculating neutrophils were highly activated in patients with KD and MIS-C and were a major source of IL-1ß. Following IVIG treatment, activated IL-1ß+ neutrophils were reduced in the circulation. In vitro, IVIG was a potent activator of neutrophil cell death via PI3K and NADPH oxidase, but independently of caspase activation.CONCLUSIONSActivated neutrophils expressing IL-1ß can be targeted by IVIG, supporting its use in both KD and MIS-C to ameliorate inflammation.FUNDINGPatient Centered Outcomes Research Institute; NIH; American Asthma Foundation; American Heart Association; Novo Nordisk Foundation; NIGMS; American Academy of Allergy, Asthma and Immunology Foundation.

Inflammasome Activation in Children With Kawasaki Disease and Multisystem Inflammatory Syndrome.

[Figure: see text].

Interactions of SARS-CoV-2 envelope protein with amilorides correlate with antiviral activity.

SARS-CoV-2 is the novel coronavirus that is the causative agent of COVID-19, a sometimes-lethal respiratory infection responsible for a world-wide pandemic. The envelope (E) protein, one of four structural proteins encoded in the viral genome, is a 75-residue integral membrane protein whose transmembrane domain exhibits ion channel activity and whose cytoplasmic domain participates in protein-protein interactions. These activities contribute to several aspects of the viral replication-cycle, including virion assembly, budding, release, and pathogenesis. Here, we describe the structure and dynamics of full-length SARS-CoV-2 E protein in hexadecylphosphocholine micelles by NMR spectroscopy. We also characterized its interactions with four putative ion channel inhibitors. The chemical shift index and dipolar wave plots establish that E protein consists of a long transmembrane helix (residues 8-43) and a short cytoplasmic helix (residues 53-60) connected by a complex linker that exhibits some internal mobility. The conformations of the N-terminal transmembrane domain and the C-terminal cytoplasmic domain are unaffected by truncation from the intact protein. The chemical shift perturbations of E protein spectra induced by the addition of the inhibitors demonstrate that the N-terminal region (residues 6-18) is the principal binding site. The binding affinity of the inhibitors to E protein in micelles correlates with their antiviral potency in Vero E6 cells: HMA ≈ EIPA > DMA >> Amiloride, suggesting that bulky hydrophobic groups in the 5' position of the amiloride pyrazine ring play essential roles in binding to E protein and in antiviral activity. An N15A mutation increased the production of virus-like particles, induced significant chemical shift changes from residues in the inhibitor binding site, and abolished HMA binding, suggesting that Asn15 plays a key role in maintaining the protein conformation near the binding site. These studies provide the foundation for complete structure determination of E protein and for structure-based drug discovery targeting this protein.

The Prolyl-tRNA Synthetase Inhibitor Halofuginone Inhibits SARS-CoV-2 Infection.

We identify the prolyl-tRNA synthetase (PRS) inhibitor halofuginone 1 , a compound in clinical trials for anti-fibrotic and anti-inflammatory applications 2 , as a potent inhibitor of SARS-CoV-2 infection and replication. The interaction of SARS-CoV-2 spike protein with cell surface heparan sulfate (HS) promotes viral entry 3 . We find that halofuginone reduces HS biosynthesis, thereby reducing spike protein binding, SARS-CoV-2 pseudotyped virus, and authentic SARS-CoV-2 infection. Halofuginone also potently suppresses SARS-CoV-2 replication post-entry and is 1,000-fold more potent than Remdesivir 4 . Inhibition of HS biosynthesis and SARS-CoV-2 infection depends on specific inhibition of PRS, possibly due to translational suppression of proline-rich proteins. We find that pp1a and pp1ab polyproteins of SARS-CoV-2, as well as several HS proteoglycans, are proline-rich, which may make them particularly vulnerable to halofuginone's translational suppression. Halofuginone is orally bioavailable, has been evaluated in a phase I clinical trial in humans and distributes to SARS-CoV-2 target organs, including the lung, making it a near-term clinical trial candidate for the treatment of COVID-19.

Multi-clonal SARS-CoV-2 neutralization by antibodies isolated from severe COVID-19 convalescent donors.

The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe, and not mild, infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of ACE2:RBD inhibition. B cell receptor (BCR) sequencing revealed that VH3-53 was enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against authentic SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and mutagenesis of RBD, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19.

Multi-Clonal Live SARS-CoV-2 In Vitro Neutralization by Antibodies Isolated from Severe COVID-19 Convalescent Donors.

The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe and not mild infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of viral inhibition. B cell receptor (BCR) sequencing revealed two VH genes, VH3-38 and VH3-53, that were enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against live SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and RBD mutagenesis, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19.

Age-dependent regulation of SARS-CoV-2 cell entry genes and cell death programs correlates with COVID-19 disease severity.

Angiotensin-converting enzyme 2 (ACE2) maintains cardiovascular and renal homeostasis but also serves as the entry receptor for the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2), the causal agent of novel coronavirus disease 2019 (COVID-19). COVID-19 disease severity is typically lower in pediatric patients than adults (particularly the elderly), but higher rates of hospitalizations requiring intensive care are observed in infants than in older children - the reasons for these differences are unknown. ACE2 is expressed in several adult tissues and cells, including alveolar type 2 cells of the distal lung epithelium, but expression at other ages is largely unexplored. Here we show that ACE2 transcripts are expressed in the lung and trachea shortly after birth, downregulated during childhood, and again expressed at high levels in late adulthood. Notably, the repertoire of cells expressing ACE2 protein in the mouse lung and airways shifts during key phases of lung maturation. In particular, podoplanin-positive cells, which are likely alveolar type I cells responsible for gas exchange, express ACE2 only in advanced age. Similar patterns of expression were evident in analysis of human lung tissue from over 100 donors, along with extreme inter- and intra-individual heterogeneity in ACE2 protein expression in epithelial cells. Furthermore, we find that apoptosis, which is a natural host defense system against viral infection, is dynamically regulated during lung maturation, resulting in periods of heightened apoptotic priming and dependence on pro-survival BCL-2 family proteins including MCL-1. Infection of human lung cells with SARS-CoV-2 triggers an unfolded protein stress response and upregulation of the endogenous MCL-1 inhibitor Noxa; in young individuals, MCL-1 inhibition is sufficient to trigger apoptosis in lung epithelial cells and may thus limit virion production and inflammatory signaling. Overall, we identify strong and distinct correlates of COVID-19 disease severity across lifespan and advance our understanding of the regulation of ACE2 and cell death programs in the mammalian lung. Furthermore, our work provides the framework for translation of apoptosis modulating drugs as novel treatments for COVID-19.

Cholesterol 25-Hydroxylase inhibits SARS-CoV-2 and other coronaviruses by depleting membrane cholesterol.

Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2 and has spread across the globe. SARS-CoV-2 is a highly infectious virus with no vaccine or antiviral therapy available to control the pandemic; therefore, it is crucial to understand the mechanisms of viral pathogenesis and the host immune responses to SARS-CoV-2. SARS-CoV-2 is a new member of the betacoronavirus genus like other closely related viruses including SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Both SARS-CoV and MERS-CoV have caused serious outbreaks and epidemics in the past eighteen years. Here, we report that one of the interferon-stimulated genes (ISGs), cholesterol 25-hydroxylase (CH25H), is induced by SARS-CoV-2 infection in vitro and in COVID-19-infected patients. CH25H converts cholesterol to 25-hydrocholesterol (25HC) and 25HC shows broad anti-coronavirus activity by blocking membrane fusion. Furthermore, 25HC inhibits USA-WA1/2020 SARS-CoV-2 infection in lung epithelial cells and viral entry in human lung organoids. Mechanistically, 25HC inhibits viral membrane fusion by activating the ER-localized acyl-CoA:cholesterol acyltransferase (ACAT) which leads to the depletion of accessible cholesterol from the plasma membrane. Altogether, our results shed light on a potentially broad antiviral mechanism by 25HC through depleting accessible cholesterol on the plasma membrane to suppress virus-cell fusion. Since 25HC is a natural product with no known toxicity at effective concentrations, it provides a potential therapeutic candidate for COVID-19 and emerging viral diseases in the future.

A Two-Cell Model for IL-1ß Release Mediated by Death-Receptor Signaling.

Interleukin-1ß (IL-1ß) is a key orchestrator of anti-microbial immunity whose secretion is typically dependent on activation of inflammasomes. However, many pathogens have evolved strategies to evade inflammasome activation. Here we describe an alternative, two-cell model for IL-1ß release where invariant natural killer T (iNKT) cells use the death receptor pathway to instruct antigen-presenting cells to secrete IL-1ß. Following cognate interactions with TLR-primed bone marrow-derived dendritic cells (BMDCs), iNKT cells rapidly translocate intracellular Fas ligand to the surface to engage Fas on BMDCs. Fas ligation activates a caspase-8-dependent signaling cascade in BMDCs that drives IL-1ß release largely independent of inflammasomes. The apoptotic program initiated by Fas ligation rapidly transitions into a pyroptosis-like form of cell death mediated by gasdermin D. Together, our findings support a two-cell model for IL-1ß secretion that may supersede inflammasome activation when cytosolic triggers fail.

Ptpn6 inhibits caspase-8- and Ripk3/Mlkl-dependent inflammation.

Ptpn6 is a cytoplasmic phosphatase that functions to prevent autoimmune and interleukin-1 (IL-1) receptor-dependent, caspase-1-independent inflammatory disease. Conditional deletion of Ptpn6 in neutrophils (Ptpn6∆PMN) is sufficient to initiate IL-1 receptor-dependent cutaneous inflammatory disease, but the source of IL-1 and the mechanisms behind IL-1 release remain unclear. Here, we investigate the mechanisms controlling IL-1α/ß release from neutrophils by inhibiting caspase-8-dependent apoptosis and Ripk1-Ripk3-Mlkl-regulated necroptosis. Loss of Ripk1 accelerated disease onset, whereas combined deletion of caspase-8 and either Ripk3 or Mlkl strongly protected Ptpn6∆PMN mice. Ptpn6∆PMN neutrophils displayed increased p38 mitogen-activated protein kinase-dependent Ripk1-independent IL-1 and tumor necrosis factor production, and were prone to cell death. Together, these data emphasize dual functions for Ptpn6 in the negative regulation of p38 mitogen-activated protein kinase activation to control tumor necrosis factor and IL-1α/ß expression, and in maintaining Ripk1 function to prevent caspase-8- and Ripk3-Mlkl-dependent cell death and concomitant IL-1α/ß release.

ß-glucan-dependent shuttling of conidia from neutrophils to macrophages occurs during fungal infection establishment.

The initial host response to fungal pathogen invasion is critical to infection establishment and outcome. However, the diversity of leukocyte-pathogen interactions is only recently being appreciated. We describe a new form of interleukocyte conidial exchange called "shuttling." In Talaromyces marneffei and Aspergillus fumigatus zebrafish in vivo infections, live imaging demonstrated conidia initially phagocytosed by neutrophils were transferred to macrophages. Shuttling is unidirectional, not a chance event, and involves alterations of phagocyte mobility, intercellular tethering, and phagosome transfer. Shuttling kinetics were fungal-species-specific, implicating a fungal determinant. ß-glucan serves as a fungal-derived signal sufficient for shuttling. Murine phagocytes also shuttled in vitro. The impact of shuttling for microbiological outcomes of in vivo infections is difficult to specifically assess experimentally, but for these two pathogens, shuttling augments initial conidial redistribution away from fungicidal neutrophils into the favorable macrophage intracellular niche. Shuttling is a frequent host-pathogen interaction contributing to fungal infection establishment patterns.