High expression of oleoyl-ACP hydrolase underpins life-threatening respiratory viral diseases.

Respiratory infections cause significant morbidity and mortality, yet it is unclear why some individuals succumb to severe disease. In patients hospitalized with avian A(H7N9) influenza, we investigated early drivers underpinning fatal disease. Transcriptomics strongly linked oleoyl-acyl-carrier-protein (ACP) hydrolase (OLAH), an enzyme mediating fatty acid production, with fatal A(H7N9) early after hospital admission, persisting until death. Recovered patients had low OLAH expression throughout hospitalization. High OLAH levels were also detected in patients hospitalized with life-threatening seasonal influenza, COVID-19, respiratory syncytial virus (RSV), and multisystem inflammatory syndrome in children (MIS-C) but not during mild disease. In olah-/- mice, lethal influenza infection led to survival and mild disease as well as reduced lung viral loads, tissue damage, infection-driven pulmonary cell infiltration, and inflammation. This was underpinned by differential lipid droplet dynamics as well as reduced viral replication and virus-induced inflammation in macrophages. Supplementation of oleic acid, the main product of OLAH, increased influenza replication in macrophages and their inflammatory potential. Our findings define how the expression of OLAH drives life-threatening viral disease.

SARS-CoV-2 infection triggers profibrotic macrophage responses and lung fibrosis.

COVID-19-induced "acute respiratory distress syndrome" (ARDS) is associated with prolonged respiratory failure and high mortality, but the mechanistic basis of lung injury remains incompletely understood. Here, we analyze pulmonary immune responses and lung pathology in two cohorts of patients with COVID-19 ARDS using functional single-cell genomics, immunohistology, and electron microscopy. We describe an accumulation of CD163-expressing monocyte-derived macrophages that acquired a profibrotic transcriptional phenotype during COVID-19 ARDS. Gene set enrichment and computational data integration revealed a significant similarity between COVID-19-associated macrophages and profibrotic macrophage populations identified in idiopathic pulmonary fibrosis. COVID-19 ARDS was associated with clinical, radiographic, histopathological, and ultrastructural hallmarks of pulmonary fibrosis. Exposure of human monocytes to SARS-CoV-2, but not influenza A virus or viral RNA analogs, was sufficient to induce a similar profibrotic phenotype in vitro. In conclusion, we demonstrate that SARS-CoV-2 triggers profibrotic macrophage responses and pronounced fibroproliferative ARDS.

Self-Recognition of an Inducible Host lncRNA by RIG-I Feedback Restricts Innate Immune Response.

The innate RNA sensor RIG-I is critical in the initiation of antiviral type I interferons (IFNs) production upon recognition of "non-self" viral RNAs. Here, we identify a host-derived, IFN-inducible long noncoding RNA, lnc-Lsm3b, that can compete with viral RNAs in the binding of RIG-I monomers and feedback inactivate the RIG-I innate function at late stage of innate response. Mechanistically, binding of lnc-Lsm3b restricts RIG-I protein's conformational shift and prevents downstream signaling, thereby terminating type I IFNs production. Multivalent structural motifs and long-stem structure are critical features of lnc-Lsm3b for RIG-I binding and inhibition. These data reveal a non-canonical self-recognition mode in the regulation of immune response and demonstrate an important role of an inducible "self" lncRNA acting as a potent molecular decoy actively saturating RIG-I binding sites to restrict the duration of "non-self" RNA-induced innate immune response and maintaining immune homeostasis, with potential utility in inflammatory disease management.

Transcription Elongation Can Affect Genome 3D Structure.

How transcription affects genome 3D organization is not well understood. We found that during influenza A (IAV) infection, rampant transcription rapidly reorganizes host cell chromatin interactions. These changes occur at the ends of highly transcribed genes, where global inhibition of transcription termination by IAV NS1 protein causes readthrough transcription for hundreds of kilobases. In these readthrough regions, elongating RNA polymerase II disrupts chromatin interactions by inducing cohesin displacement from CTCF sites, leading to locus decompaction. Readthrough transcription into heterochromatin regions switches them from the inert (B) to the permissive (A) chromatin compartment and enables transcription factor binding. Data from non-viral transcription stimuli show that transcription similarly affects cohesin-mediated chromatin contacts within gene bodies. Conversely, inhibition of transcription elongation allows cohesin to accumulate at previously transcribed intragenic CTCF sites and to mediate chromatin looping and compaction. Our data indicate that transcription elongation by RNA polymerase II remodels genome 3D architecture.

A Long Noncoding RNA lincRNA-EPS Acts as a Transcriptional Brake to Restrain Inflammation.

Long intergenic noncoding RNAs (lincRNAs) are important regulators of gene expression. Although lincRNAs are expressed in immune cells, their functions in immunity are largely unexplored. Here, we identify an immunoregulatory lincRNA, lincRNA-EPS, that is precisely regulated in macrophages to control the expression of immune response genes (IRGs). Transcriptome analysis of macrophages from lincRNA-EPS-deficient mice, combined with gain-of-function and rescue experiments, revealed a specific role for this lincRNA in restraining IRG expression. Consistently, lincRNA-EPS-deficient mice manifest enhanced inflammation and lethality following endotoxin challenge in vivo. lincRNA-EPS localizes at regulatory regions of IRGs to control nucleosome positioning and repress transcription. Further, lincRNA-EPS mediates these effects by interacting with heterogeneous nuclear ribonucleoprotein L via a CANACA motif located in its 3' end. Together, these findings identify lincRNA-EPS as a repressor of inflammatory responses, highlighting the importance of lincRNAs in the immune system.

Resistance of HIV-infected macrophages to CD8+ T lymphocyte-mediated killing drives activation of the immune system.

CD4+ T lymphocytes are the principal target of human immunodeficiency virus (HIV), but infected macrophages also contribute to viral pathogenesis. The killing of infected cells by CD8+ cytotoxic T lymphocytes (CTLs) leads to control of viral replication. Here we found that the killing of macrophages by CTLs was impaired relative to the killing of CD4+ T cells by CTLs, and this resulted in inefficient suppression of HIV. The killing of macrophages depended on caspase-3 and granzyme B, whereas the rapid killing of CD4+ T cells was caspase independent and did not require granzyme B. Moreover, the impaired killing of macrophages was associated with prolonged effector cell-target cell contact time and higher expression of interferon-γ by CTLs, which induced macrophage production of pro-inflammatory chemokines that recruited monocytes and T cells. Similar results were obtained when macrophages presented other viral antigens, suggestive of a general mechanism for macrophage persistence as antigen-presenting cells that enhance inflammation and adaptive immunity. Inefficient killing of macrophages by CTLs might contribute to chronic inflammation, a hallmark of chronic disease caused by HIV.

Nuclear RNF2 inhibits interferon function by promoting K33-linked STAT1 disassociation from DNA.

Prolonged activation of interferon-STAT1 signaling is closely related to inflammatory autoimmune disorders, and therefore the identification of negative regulators of these pathways is important. Through high-content screening of 115 mouse RING-domain E3 ligases, we identified the E3 ubiquitin ligase RNF2 as a potent inhibitor of interferon-dependent antiviral responses. RNF2 deficiency substantially enhanced interferon-stimulated gene (ISG) expression and antiviral responses. Mechanistically, nuclear RNF2 directly bound to STAT1 after interferon stimulation and increased K33-linked polyubiquitination of the DNA-binding domain of STAT1 at position K379, in addition to promoting the disassociation of STAT1/STAT2 from DNA and consequently suppressing ISG transcription. Our study provides insight into the regulation of interferon-dependent responses via a previously unrecognized post-translational modification of STAT1 in the nucleus.

Phosphatidylserine vesicles enable efficient en bloc transmission of enteroviruses.

A central paradigm within virology is that each viral particle largely behaves as an independent infectious unit. Here, we demonstrate that clusters of enteroviral particles are packaged within phosphatidylserine (PS) lipid-enriched vesicles that are non-lytically released from cells and provide greater infection efficiency than free single viral particles. We show that vesicular PS lipids are co-factors to the relevant enterovirus receptors in mediating subsequent infectivity and transmission, in particular to primary human macrophages. We demonstrate that clustered packaging of viral particles within vesicles enables multiple viral RNA genomes to be collectively transferred into single cells. This study reveals a novel mode of viral transmission, where enteroviral genomes are transmitted from cell-to-cell en bloc in membrane-bound PS vesicles instead of as single independent genomes. This has implications for facilitating genetic cooperativity among viral quasispecies as well as enhancing viral replication.

An aberrant immune-epithelial progenitor niche drives viral lung sequelae.

The long-term physiological consequences of respiratory viral infections, particularly in the aftermath of the COVID-19 pandemic-termed post-acute sequelae of SARS-CoV-2 (PASC)-are rapidly evolving into a major public health concern1-3. While the cellular and molecular aetiologies of these sequelae are poorly defined, increasing evidence implicates abnormal immune responses3-6 and/or impaired organ recovery7-9 after infection. However, the precise mechanisms that link these processes in the context of PASC remain unclear. Here, with insights from three cohorts of patients with respiratory PASC, we established a mouse model of post-viral lung disease and identified an aberrant immune-epithelial progenitor niche unique to fibroproliferation in respiratory PASC. Using spatial transcriptomics and imaging, we found a central role for lung-resident CD8+ T cell-macrophage interactions in impairing alveolar regeneration and driving fibrotic sequelae after acute viral pneumonia. Specifically, IFNγ and TNF derived from CD8+ T cells stimulated local macrophages to chronically release IL-1ß, resulting in the long-term maintenance of dysplastic epithelial progenitors and lung fibrosis. Notably, therapeutic neutralization of IFNγ + TNF or IL-1ß markedly improved alveolar regeneration and pulmonary function. In contrast to other approaches, which require early intervention10, we highlight therapeutic strategies to rescue fibrotic disease after the resolution of acute disease, addressing a current unmet need in the clinical management of PASC and post-viral disease.

Human SARS-CoV-2 challenge uncovers local and systemic response dynamics.

The COVID-19 pandemic is an ongoing global health threat, yet our understanding of the dynamics of early cellular responses to this disease remains limited1. Here in our SARS-CoV-2 human challenge study, we used single-cell multi-omics profiling of nasopharyngeal swabs and blood to temporally resolve abortive, transient and sustained infections in seronegative individuals challenged with pre-Alpha SARS-CoV-2. Our analyses revealed rapid changes in cell-type proportions and dozens of highly dynamic cellular response states in epithelial and immune cells associated with specific time points and infection status. We observed that the interferon response in blood preceded the nasopharyngeal response. Moreover, nasopharyngeal immune infiltration occurred early in samples from individuals with only transient infection and later in samples from individuals with sustained infection. High expression of HLA-DQA2 before inoculation was associated with preventing sustained infection. Ciliated cells showed multiple immune responses and were most permissive for viral replication, whereas nasopharyngeal T cells and macrophages were infected non-productively. We resolved 54 T cell states, including acutely activated T cells that clonally expanded while carrying convergent SARS-CoV-2 motifs. Our new computational pipeline Cell2TCR identifies activated antigen-responding T cells based on a gene expression signature and clusters these into clonotype groups and motifs. Overall, our detailed time series data can serve as a Rosetta stone for epithelial and immune cell responses and reveals early dynamic responses associated with protection against infection.

HIV-1 blocks the signaling adaptor MAVS to evade antiviral host defense after sensing of abortive HIV-1 RNA by the host helicase DDX3.

The mechanisms by which human immunodeficiency virus 1 (HIV-1) avoids immune surveillance by dendritic cells (DCs), and thereby prevents protective adaptive immune responses, remain poorly understood. Here we showed that HIV-1 actively arrested antiviral immune responses by DCs, which contributed to efficient HIV-1 replication in infected individuals. We identified the RNA helicase DDX3 as an HIV-1 sensor that bound abortive HIV-1 RNA after HIV-1 infection and induced DC maturation and type I interferon responses via the signaling adaptor MAVS. Notably, HIV-1 recognition by the C-type lectin receptor DC-SIGN activated the mitotic kinase PLK1, which suppressed signaling downstream of MAVS, thereby interfering with intrinsic host defense during HIV-1 infection. Finally, we showed that PLK1-mediated suppression of DDX3-MAVS signaling was a viral strategy that accelerated HIV-1 replication in infected individuals.

The immune response to RNA suppresses nucleic acid synthesis by limiting ribose 5-phosphate.

During infection viruses hijack host cell metabolism to promote their replication. Here, analysis of metabolite alterations in macrophages exposed to poly I:C recognises that the antiviral effector Protein Kinase RNA-activated (PKR) suppresses glucose breakdown within the pentose phosphate pathway (PPP). This pathway runs parallel to central glycolysis and is critical to producing NADPH and pentose precursors for nucleotides. Changes in metabolite levels between wild-type and PKR-ablated macrophages show that PKR controls the generation of ribose 5-phosphate, in a manner distinct from its established function in gene expression but dependent on its kinase activity. PKR phosphorylates and inhibits the Ribose 5-Phosphate Isomerase A (RPIA), thereby preventing interconversion of ribulose- to ribose 5-phosphate. This activity preserves redox control but decreases production of ribose 5-phosphate for nucleotide biosynthesis. Accordingly, the PKR-mediated immune response to RNA suppresses nucleic acid production. In line, pharmacological targeting of the PPP during infection decreases the replication of the Herpes simplex virus. These results identify an immune response-mediated control of host cell metabolism and suggest targeting the RPIA as a potential innovative antiviral treatment.

Interaction of chikungunya virus glycoproteins with macrophage factors controls virion production.

Despite their role as innate sentinels, macrophages can serve as cellular reservoirs of chikungunya virus (CHIKV), a highly-pathogenic arthropod-borne alphavirus that has caused large outbreaks among human populations. Here, with the use of viral chimeras and evolutionary selection analysis, we define CHIKV glycoproteins E1 and E2 as critical for virion production in THP-1 derived human macrophages. Through proteomic analysis and functional validation, we further identify signal peptidase complex subunit 3 (SPCS3) and eukaryotic translation initiation factor 3 subunit K (eIF3k) as E1-binding host proteins with anti-CHIKV activities. We find that E1 residue V220, which has undergone positive selection, is indispensable for CHIKV production in macrophages, as its mutation attenuates E1 interaction with the host restriction factors SPCS3 and eIF3k. Finally, we show that the antiviral activity of eIF3k is translation-independent, and that CHIKV infection promotes eIF3k translocation from the nucleus to the cytoplasm, where it associates with SPCS3. These functions of CHIKV glycoproteins late in the viral life cycle provide a new example of an intracellular evolutionary arms race with host restriction factors, as well as potential targets for therapeutic intervention.

Identification of a role for TRIM29 in the control of innate immunity in the respiratory tract.

The respiratory tract is heavily populated with innate immune cells, but the mechanisms that control such cells are poorly defined. Here we found that the E3 ubiquitin ligase TRIM29 was a selective regulator of the activation of alveolar macrophages, the expression of type I interferons and the production of proinflammatory cytokines in the lungs. We found that deletion of TRIM29 enhanced macrophage production of type I interferons and protected mice from infection with influenza virus, while challenge of Trim29-/- mice with Haemophilus influenzae resulted in lethal lung inflammation due to massive production of proinflammatory cytokines by macrophages. Mechanistically, we demonstrated that TRIM29 inhibited interferon-regulatory factors and signaling via the transcription factor NF-κB by degrading the adaptor NEMO and that TRIM29 directly bound NEMO and subsequently induced its ubiquitination and proteolytic degradation. These data identify TRIM29 as a key negative regulator of alveolar macrophages and might have important clinical implications for local immunity and immunopathology.

Methyltransferase Dnmt3a upregulates HDAC9 to deacetylate the kinase TBK1 for activation of antiviral innate immunity.

The DNA methyltransferase Dnmt3a has high expression in terminally differentiated macrophages; however, its role in innate immunity remains unknown. Here we report that deficiency in Dnmt3a selectively impaired the production of type I interferons triggered by pattern-recognition receptors (PRRs), but not that of the proinflammatory cytokines TNF and IL-6. Dnmt3a-deficient mice exhibited enhanced susceptibility to viral challenge. Dnmt3a did not directly regulate the transcription of genes encoding type I interferons; instead, it increased the production of type I interferons through an epigenetic mechanism by maintaining high expression of the histone deacetylase HDAC9. In turn, HDAC9 directly maintained the deacetylation status of the key PRR signaling molecule TBK1 and enhanced its kinase activity. Our data add mechanistic insight into the crosstalk between epigenetic modifications and post-translational modifications in the regulation of PRR signaling and activation of antiviral innate immune responses.

Inflammasome activation in infected macrophages drives COVID-19 pathology.

Severe COVID-19 is characterized by persistent lung inflammation, inflammatory cytokine production, viral RNA and a sustained interferon (IFN) response, all of which are recapitulated and required for pathology in the SARS-CoV-2-infected MISTRG6-hACE2 humanized mouse model of COVID-19, which has a human immune system1-20. Blocking either viral replication with remdesivir21-23 or the downstream IFN-stimulated cascade with anti-IFNAR2 antibodies in vivo in the chronic stages of disease attenuates the overactive immune inflammatory response, especially inflammatory macrophages. Here we show that SARS-CoV-2 infection and replication in lung-resident human macrophages is a critical driver of disease. In response to infection mediated by CD16 and ACE2 receptors, human macrophages activate inflammasomes, release interleukin 1 (IL-1) and IL-18, and undergo pyroptosis, thereby contributing to the hyperinflammatory state of the lungs. Inflammasome activation and the accompanying inflammatory response are necessary for lung inflammation, as inhibition of the NLRP3 inflammasome pathway reverses chronic lung pathology. Notably, this blockade of inflammasome activation leads to the release of infectious virus by the infected macrophages. Thus, inflammasomes oppose host infection by SARS-CoV-2 through the production of inflammatory cytokines and suicide by pyroptosis to prevent a productive viral cycle.

The receptor TREML4 amplifies TLR7-mediated signaling during antiviral responses and autoimmunity.

The molecules and pathways that fine-tune innate inflammatory responses mediated by Toll-like receptor 7 (TLR7) remain to be fully elucidated. Using an unbiased genome-scale screen with short hairpin RNA (shRNA), we identified the receptor TREML4 as an essential positive regulator of TLR7 signaling. Macrophages from Treml4(-/-) mice were hyporesponsive to TLR7 agonists and failed to produce type I interferons due to impaired phosphorylation of the transcription factor STAT1 by the mitogen-activated protein kinase p38 and decreased recruitment of the adaptor MyD88 to TLR7. TREML4 deficiency reduced the production of inflammatory cytokines and autoantibodies in MRL/lpr mice, which are prone to systemic lupus erythematosus (SLE), and inhibited the antiviral immune response to influenza virus. Our data identify TREML4 as a positive regulator of TLR7 signaling and provide insight into the molecular mechanisms that control antiviral immunity and the development of autoimmunity.

A molecular single-cell lung atlas of lethal COVID-19.

Respiratory failure is the leading cause of death in patients with severe SARS-CoV-2 infection1,2, but the host response at the lung tissue level is poorly understood. Here we performed single-nucleus RNA sequencing of about 116,000 nuclei from the lungs of nineteen individuals who died of COVID-19 and underwent rapid autopsy and seven control individuals. Integrated analyses identified substantial alterations in cellular composition, transcriptional cell states, and cell-to-cell interactions, thereby providing insight into the biology of lethal COVID-19. The lungs from individuals with COVID-19 were highly inflamed, with dense infiltration of aberrantly activated monocyte-derived macrophages and alveolar macrophages, but had impaired T cell responses. Monocyte/macrophage-derived interleukin-1ß and epithelial cell-derived interleukin-6 were unique features of SARS-CoV-2 infection compared to other viral and bacterial causes of pneumonia. Alveolar type 2 cells adopted an inflammation-associated transient progenitor cell state and failed to undergo full transition into alveolar type 1 cells, resulting in impaired lung regeneration. Furthermore, we identified expansion of recently described CTHRC1+ pathological fibroblasts3 contributing to rapidly ensuing pulmonary fibrosis in COVID-19. Inference of protein activity and ligand-receptor interactions identified putative drug targets to disrupt deleterious circuits. This atlas enables the dissection of lethal COVID-19, may inform our understanding of long-term complications of COVID-19 survivors, and provides an important resource for therapeutic development.

BUD13 Promotes a Type I Interferon Response by Countering Intron Retention in Irf7.

Intron retention (IR) has emerged as an important mechanism of gene expression control, but the factors controlling IR events remain poorly understood. We observed consistent IR in one intron of the Irf7 gene and identified BUD13 as an RNA-binding protein that acts at this intron to increase the amount of successful splicing. Deficiency in BUD13 was associated with increased IR, decreased mature Irf7 transcript and protein levels, and consequently a dampened type I interferon response, which compromised the ability of BUD13-deficient macrophages to withstand vesicular stomatitis virus (VSV) infection. Global analysis of BUD13 knockdown and BUD13 cross-linking to RNA revealed a subset of introns that share many characteristics with the one found in Irf7 and are spliced in a BUD13-dependent manner. Deficiency of BUD13 led to decreased mature transcript from genes containing such introns. Thus, by acting as an antagonist to IR, BUD13 facilitates the expression of genes at which IR occurs.

Monocyte to macrophage differentiation and changes in cellular redox homeostasis promote cell type-specific HIV latency reactivation.

HIV latency regulation in monocytes and macrophages can vary according to signals directing differentiation, polarization, and function. To investigate these processes, we generated an HIV latency model in THP-1 monocytes and showed differential levels of HIV reactivation among clonal populations. Monocyte-to-macrophage differentiation of HIV-infected primary human CD14+ and THP-1 cells induced HIV reactivation and showed that virus production increased concomitant with macrophage differentiation. We applied the HIV-infected THP-1 monocyte-to-macrophage (MLat) model to assess the biological mechanisms regulating HIV latency dynamics during monocyte-to-macrophage differentiation. We pinpointed protein kinase C signaling pathway activation and Cyclin T1 upregulation as inherent differentiation mechanisms that regulate HIV latency reactivation. Macrophage polarization regulated latency, revealing proinflammatory M1 macrophages suppressed HIV reactivation while anti-inflammatory M2 macrophages promoted HIV reactivation. Because macrophages rely on reactive-oxygen species (ROS) to exert numerous cellular functions, we disrupted redox pathways and found that inhibitors of the thioredoxin (Trx) system acted as latency-promoting agents in T-cells and monocytes, but opposingly acted as latency-reversing agents in macrophages. We explored this mechanism with Auranofin, a clinical candidate for reducing HIV reservoirs, and demonstrated Trx reductase inhibition led to ROS induced NF-κB activity, which promoted HIV reactivation in macrophages, but not in T-cells and monocytes. Collectively, cell type-specific differences in HIV latency regulation could pose a barrier to HIV eradication strategies.