Truncating NFKB1 variants cause combined NLRP3 inflammasome activation and type I interferon signaling and predispose to necrotizing fasciitis.

In monogenic autoinflammatory diseases, mutations in genes regulating innate immune responses often lead to uncontrolled activation of inflammasome pathways or the type I interferon (IFN-I) response. We describe a mechanism of autoinflammation potentially predisposing patients to life-threatening necrotizing soft tissue inflammation. Six unrelated families are identified in which affected members present with necrotizing fasciitis or severe soft tissue inflammations. Exome sequencing reveals truncating monoallelic loss-of-function variants of nuclear factor κ light-chain enhancer of activated B cells (NFKB1) in affected patients. In patients' macrophages and in NFKB1-variant-bearing THP-1 cells, activation increases both interleukin (IL)-1ß secretion and IFN-I signaling. Truncation of NF-κB1 impairs autophagy, accompanied by the accumulation of reactive oxygen species and reduced degradation of inflammasome receptor nucleotide-binding oligomerization domain, leucine-rich repeat-containing protein 3 (NLRP3), and Toll/IL-1 receptor domain-containing adaptor protein inducing IFN-ß (TRIF), thus leading to combined excessive inflammasome and IFN-I activity. Many of the patients respond to anti-inflammatory treatment, and targeting IL-1ß and/or IFN-I signaling could represent a therapeutic approach for these patients.

Lipoprotein(a) induces caspase-1 activation and IL-1 signaling in human macrophages.

Introduction: Lipoprotein(a) (Lp(a)) is an LDL-like particle with an additional apolipoprotein (apo)(a) covalently attached. Elevated levels of circulating Lp(a) are a risk factor for atherosclerosis. A proinflammatory role for Lp(a) has been proposed, but its molecular details are incompletely defined. Methods and results: To explore the effect of Lp(a) on human macrophages we performed RNA sequencing on THP-1 macrophages treated with Lp(a) or recombinant apo(a), which showed that especially Lp(a) induces potent inflammatory responses. Thus, we stimulated THP-1 macrophages with serum containing various Lp(a) levels to investigate their correlations with cytokines highlighted by the RNAseq, showing significant correlations with caspase-1 activity and secretion of IL-1ß and IL-18. We further isolated both Lp(a) and LDL particles from three donors and then compared their atheroinflammatory potentials together with recombinant apo(a) in primary and THP-1 derived macrophages. Compared with LDL, Lp(a) induced a robust and dose-dependent caspase-1 activation and release of IL-1ß and IL-18 in both macrophage types. Recombinant apo(a) strongly induced caspase-1 activation and IL-1ß release in THP-1 macrophages but yielded weak responses in primary macrophages. Structural analysis of these particles revealed that the Lp(a) proteome was enriched in proteins associated with complement activation and coagulation, and its lipidome was relatively deficient in polyunsaturated fatty acids and had a high n-6/n-3 ratio promoting inflammation. Discussion: Our data show that Lp(a) particles induce the expression of inflammatory genes, and Lp(a) and to a lesser extent apo(a) induce caspase-1 activation and IL-1 signaling. Major differences in the molecular profiles between Lp(a) and LDL contribute to Lp(a) being more atheroinflammatory.

Lipopolysaccharide Primes Human Macrophages for Noncanonical Inflammasome-Induced Extracellular Vesicle Secretion.

Human macrophages secrete extracellular vesicles (EVs) loaded with numerous immunoregulatory proteins. Vesicle-mediated protein secretion in macrophages is regulated by poorly characterized mechanisms; however, it is now known that inflammatory conditions significantly alter both the quantities and protein composition of secreted vesicles. In this study, we employed high-throughput quantitative proteomics to characterize the modulation of EV-mediated protein secretion during noncanonical caspase-4/5 inflammasome activation via LPS transfection. We show that human macrophages activate robust caspase-4-dependent EV secretion upon transfection of LPS, and this process is also partially dependent on NLRP3 and caspase-5. A similar effect occurs with delivery of the LPS with Escherichia coli-derived outer membrane vesicles. Moreover, sensitization of the macrophages through TLR4 by LPS priming prior to LPS transfection dramatically augments the EV-mediated protein secretion. Our data demonstrate that this process differs significantly from canonical inflammasome activator ATP-induced vesiculation, and it is dependent on the autocrine IFN signal associated with TLR4 activation. LPS priming preceding the noncanonical inflammasome activation significantly enhances vesicle-mediated secretion of inflammasome components caspase-1, ASC, and lytic cell death effectors GSDMD, MLKL, and NINJ1, suggesting that inflammatory EV transfer may exert paracrine effects in recipient cells. Moreover, using bioinformatics methods, we identify 15-deoxy-Δ12,14-PGJ2 and parthenolide as inhibitors of caspase-4-mediated inflammation and vesicle secretion, indicating new therapeutic potential of these anti-inflammatory drugs.

Inflammasomes and SARS-CoV-2 Infection.

SARS-CoV-2 is a new type of coronavirus that has caused worldwide pandemic. The disease induced by SARS-CoV-2 is called COVID-19. A majority of people with COVID-19 have relatively mild respiratory symptoms. However, a small percentage of COVID-19 patients develop a severe disease where multiple organs are affected. These severe forms of SARS-CoV-2 infections are associated with excessive production of pro-inflammatory cytokines, so called "cytokine storm". Inflammasomes, which are protein complexes of the innate immune system orchestrate development of local and systemic inflammation during virus infection. Recent data suggest involvement of inflammasomes in severe COVID-19. Activation of inflammasome exerts two major effects: it activates caspase-1-mediated processing and secretion of pro-inflammatory cytokines IL-1ß and IL-18, and induces inflammatory cell death, pyroptosis, via protein called gasdermin D. Here, we provide comprehensive review of current understanding of the activation and possible functions of different inflammasome structures during SARS-CoV-2 infection and compare that to response caused by influenza A virus. We also discuss how novel SARS-CoV-2 mRNA vaccines activate innate immune response, which is a prerequisite for the activation of protective adaptive immune response.

Native and oxidised lipoproteins negatively regulate the serum amyloid A-induced NLRP3 inflammasome activation in human macrophages.

OBJECTIVES: The NLRP3 inflammasome plays a key role in arterial wall inflammation. In this study, we elucidated the role of serum lipoproteins in the regulation of NLRP3 inflammasome activation by serum amyloid A (SAA) and other inflammasome activators. METHODS: The effect of lipoproteins on the NLRP3 inflammasome activation was studied in primary human macrophages and THP-1 macrophages. The effect of oxidised low-density lipoprotein (LDL) was examined in an in vivo mouse model of SAA-induced peritoneal inflammation. RESULTS: Native and oxidised high-density lipoproteins (HDL3) and LDLs inhibited the interaction of SAA with TLR4. HDL3 and LDL inhibited the secretion of interleukin (IL)-1ß and tumor necrosis factor by reducing their transcription. Oxidised forms of these lipoproteins reduced the secretion of mature IL-1ß also by inhibiting the activation of NLRP3 inflammasome induced by SAA, ATP, nigericin and monosodium urate crystals. Specifically, oxidised LDL was found to inhibit the inflammasome complex formation. No cellular uptake of lipoproteins was required, nor intact lipoprotein particles for the inhibitory effect, as the lipid fraction of oxidised LDL was sufficient. The inhibition of NLRP3 inflammasome activation by oxidised LDL was partially dependent on autophagy. Finally, oxidised LDL inhibited the SAA-induced peritoneal inflammation and IL-1ß secretion in vivo. CONCLUSIONS: These findings reveal that both HDL3 and LDL inhibit the proinflammatory activity of SAA and this inhibition is further enhanced by lipoprotein oxidation. Thus, lipoproteins possess major anti-inflammatory functions that hinder the NLRP3 inflammasome-activating signals, particularly those exerted by SAA, which has important implications in the pathogenesis of cardiovascular diseases.

Inflammasomes: Exosomal miRNAs loaded for action.

Inflammasomes are multiprotein complexes of the innate immune system. Their activation leads to robust secretion of exosomes. In this issue, Wozniak et al. (2020. J. Cell Biol. https://doi.org/10.1083/jcb.201912074) reveal a connection between inflammasome-mediated cleavage of Rab-interacting lysosomal protein (RILP) and sequence-specific loading of miRNAs into exosomes.

Function and Regulation of Noncanonical Caspase-4/5/11 Inflammasome.

Inflammasomes are multiprotein complexes of the innate immune system that orchestrate development of inflammation by activating the secretion of proinflammatory cytokines, IL-1ß and IL-18. The LPS of Gram-negative bacteria have been shown to activate a novel, noncanonical inflammasome by directly binding in the cytosol to human caspase-4 and mouse caspase-11. Activation of noncanonical inflammasome exerts two major effects: it activates the NLRP3-caspase-1-mediated processing and secretion of IL-1ß and IL-18 and induces the inflammatory cell death, pyroptosis, via gasdermin D. This previously unexpected cytosolic LPS sensing of the innate immune system provides critical hints for host response to Gram-negative bacterial infections and development of different inflammatory diseases. However, many of its molecular regulatory mechanisms are yet to be discovered. In this review, we provide comprehensive analysis of current understanding of intracellular LPS detection and pyroptosis via noncanonical inflammasome and discuss the recently proposed mechanisms of its function and regulation.

From Inflammasome to Exosome-Does Extracellular Vesicle Secretion Constitute an Inflammasome-Dependent Immune Response?

Inflammasomes are intracellular protein complexes of pattern recognition receptors and caspase-1, with essential functions in regulating inflammatory responses of macrophages and dendritic cells. The primary role of inflammasomes is to catalyze processing and secretion of pro-inflammatory cytokines IL-1ß and IL-18. Recently, intracellular non-canonical inflammasome activation by caspases-4/5, which are also regulators of pyroptosis via processing gasdermin D, has been elucidated. Caspase-1, the effector protease of inflammasome complex, is also known to modulate secretion of large number of other proteins. Thereby, besides its known role in processing pro-inflammatory cytokines, the inflammasome turns into a universal regulator of protein secretion, which allows the danger-exposed cells to release various proteins in order to alert and guide neighboring cells. Majority of these proteins are not secreted through the conventional ER-Golgi secretory pathway. Instead, they are segregated in membrane-enclosed compartment and secreted in nanosized extracellular vesicles, which protect their cargo and guide it for delivery. Growing evidence indicates that inflammasome activity correlates with enhanced secretion of extracellular vesicles and modulation of their protein cargo. This inflammasome-driven unconventional, vesicle-mediated secretion of multitude of immunoregulatory proteins may constitute a novel paradigm in inflammatory responses. In this mini review we discuss the current knowledge and highlight unsolved questions about metabolic processes, signals, and mechanisms linking inflammasome activity with regulated extracellular vesicle secretion of proteins. Further investigations on this relationship may in the future help understanding the significance of extracellular vesicle secretion in inflammatory diseases such as atherosclerosis, gouty arthritis, asthma, Alzheimer's and many others.

Proteomics to study macrophage response to viral infection.

Viral infections are a major burden to human and animal health. Immune response against viruses consists of innate and adaptive immunity which are both critical for the eradication of the viral infection. The innate immune system is the first line of defense against viral infections. Proper innate immune response is required for the activation of adaptive, humoral and cell-mediated immunity. Macrophages are innate immune cells which have a central role in detecting viral infections including influenza A and human immunodeficiency viruses. Macrophages and other host cells respond to viral infection by modulating their protein expression levels, proteins' posttranslational modifications, as well as proteins' intracellular localization and secretion. Therefore the detailed characterization how viruses dynamically manipulate host proteome is needed for understanding the molecular mechanisms of viral infection. It is critical to identify cellular host factors which are exploited by different viruses, and which are less prone for mutations and could serve as potential targets for novel antiviral compounds. Here, we review how proteomics studies have enhanced our understanding of macrophage response to viral infection with special focus on Influenza A and Human immunodeficiency viruses, and virus infections of swine. SIGNIFICANCE: Influenza A viruses (IAVs) and human immunodeficiency viruses (HIV) infect annually millions of people worldwide and they form a severe threat to human health. Both IAVs and HIV-1 can efficiently antagonize host response and develop drug-resistant variants. Most current antiviral drugs are directed against viral proteins, and there is a constant need to develop new next-generation drugs targeting host proteins that are essential for viral replication. Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are economically important swine pathogens. Both PRRSV and PCV2 cause severe respiratory tract illnesses in swine. IAVs, HIV-1, and swine viruses infect macrophages activating antiviral response against these viruses. Macrophages also have a central role in the replication and spread of these viruses. However, macrophage response to these viruses is incompletely understood. Current proteomics methods can provide a global view of host-response to viral infection which is needed for in-depth understanding the molecular mechanisms of viral infection. Here we review the current proteomics studies on macrophage response to viral infection and provide insight into the global host proteome changes upon viral infection.

Antiviral Properties of Chemical Inhibitors of Cellular Anti-Apoptotic Bcl-2 Proteins.

Viral diseases remain serious threats to public health because of the shortage of effective means of control. To combat the surge of viral diseases, new treatments are urgently needed. Here we show that small-molecules, which inhibit cellular anti-apoptotic Bcl-2 proteins (Bcl-2i), induced the premature death of cells infected with different RNA or DNA viruses, whereas, at the same concentrations, no toxicity was observed in mock-infected cells. Moreover, these compounds limited viral replication and spread. Surprisingly, Bcl-2i also induced the premature apoptosis of cells transfected with viral RNA or plasmid DNA but not of mock-transfected cells. These results suggest that Bcl-2i sensitizes cells containing foreign RNA or DNA to apoptosis. A comparison of the toxicity, antiviral activity, and side effects of six Bcl-2i allowed us to select A-1155463 as an antiviral lead candidate. Thus, our results pave the way for the further development of Bcl-2i for the prevention and treatment of viral diseases.

Proprotein convertase Furin1 expression in the Drosophila fat body is essential for a normal antimicrobial peptide response and bacterial host defense.

Invading pathogens provoke robust innate immune responses in Dipteran insects, such as Drosophila melanogaster In a systemic bacterial infection, a humoral response is induced in the fat body. Gram-positive bacteria trigger the Toll signaling pathway, whereas gram-negative bacterial infections are signaled via the immune deficiency (IMD) pathway. We show here that the RNA interference-mediated silencing of Furin1-a member of the proprotein convertase enzyme family-specifically in the fat body, results in a reduction in the expression of antimicrobial peptides. This, in turn, compromises the survival of adult fruit flies in systemic infections that are caused by both gram-positive and -negative bacteria. Furin1 plays a nonredundant role in the regulation of immune responses, as silencing of Furin2, the other member of the enzyme family, had no effect on survival or the expression of antimicrobial peptides upon a systemic infection. Furin1 does not directly affect the Toll or IMD signaling pathways, but the reduced expression of Furin1 up-regulates stress response factors in the fat body. We also demonstrate that Furin1 is a negative regulator of the Janus kinase/signal transducer and activator of transcription signaling pathway, which is implicated in stress responses in the fly. In summary, our data identify Furin1 as a novel regulator of humoral immunity and cellular stress responses in Drosophila-Aittomäki, S., Valanne, S., Lehtinen, T., Matikainen, S., Nyman, T. A., Rämet, M., Pesu, M. Proprotein convertase Furin1 expression in the Drosophila fat body is essential for a normal antimicrobial peptide response and bacterial host defense.

Mass spectrometry-based proteomic exploration of the human immune system: focus on the inflammasome, global protein secretion, and T cells.

INTRODUCTION: The immune system is our defense system against microbial infections and tissue injury, and understanding how it works in detail is essential for developing drugs for different diseases. Mass spectrometry-based proteomics can provide in-depth information on the molecular mechanisms involved in immune responses. Areas covered: Summarized are the key immunology findings obtained with MS-based proteomics in the past five years, with a focus on inflammasome activation, global protein secretion, mucosal immunology, immunopeptidome and T cells. Special focus is on extracellular vesicle-mediated protein secretion and its role in immune responses. Expert commentary: Proteomics is an essential part of modern omics-scale immunology research. To date, MS-based proteomics has been used in immunology to study protein expression levels, their subcellular localization, secretion, post-translational modifications, and interactions in immune cells upon activation by different stimuli. These studies have made major contributions to understanding the molecular mechanisms involved in innate and adaptive immune responses. New developments in proteomics offer constantly novel possibilities for exploring the immune system. Examples of these techniques include mass cytometry and different MS-based imaging approaches which can be widely used in immunology.

Global Characterization of Protein Secretion from Human Macrophages Following Non-canonical Caspase-4/5 Inflammasome Activation.

Gram-negative bacteria are associated with a wide spectrum of infectious diseases in humans. Inflammasomes are cytosolic protein complexes that are assembled when the cell encounters pathogens or other harmful agents. The non-canonical caspase-4/5 inflammasome is activated by Gram-negative bacteria-derived lipopolysaccharide (LPS) and by endogenous oxidized phospholipids. Protein secretion is a critical component of the innate immune response. Here, we have used label-free quantitative proteomics to characterize global protein secretion in response to non-canonical inflammasome activation upon intracellular LPS recognition in human primary macrophages. Before proteomics, the total secretome was separated into two fractions, enriched extracellular vesicle (EV) fraction and rest-secretome (RS) fraction using size-exclusion centrifugation. We identified 1048 proteins from the EV fraction and 1223 proteins from the RS fraction. From these, 640 were identified from both fractions suggesting that the non-canonical inflammasome activates multiple, partly overlapping protein secretion pathways. We identified several secreted proteins that have a critical role in host response against severe Gram-negative bacterial infection. The soluble secretome (RS fraction) was highly enriched with inflammation-associated proteins upon intracellular LPS recognition. Several ribosomal proteins were highly abundant in the EV fraction upon infection, and our data strongly suggest that secretion of translational machinery and concomitant inhibition of translation are important parts of host response against Gram-negative bacteria sensing caspase-4/5 inflammasome. Intracellular recognition of LPS resulted in the secretion of two metalloproteinases, adisintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and MMP14, in the enriched EV fraction. ADAM10 release was associated with the secretion of TNF, a key inflammatory cytokine, and M-CSF, an important growth factor for myeloid cells probably through ADAM10-dependent membrane shedding of these cytokines. Caspase-4/5 inflammasome activation also resulted in secretion of danger-associated molecules S100A8 and prothymosin-α in the enriched EV fraction. Both S100A8 and prothymosin-α are ligands for toll-like receptor 4 recognizing extracellular LPS, and they may contribute to endotoxic shock during non-canonical inflammasome activation.

Proteomic and Bioinformatic Characterization of Extracellular Vesicles Released from Human Macrophages upon Influenza A Virus Infection.

Influenza A viruses (IAVs) are aggressive pathogens that cause acute respiratory diseases and annual epidemics in humans. Host defense against IAV infection is initiated by macrophages, which are the principal effector cells of the innate immune system. We have previously shown that IAV infection of human macrophages is associated with robust secretion of proteins via conventional and unconventional protein release pathways. Here we have characterized unconventional, extracellular vesicle (EV)-mediated protein secretion in human macrophages during IAV infection using proteomics, bioinformatics, and functional studies. We demonstrate that at 9 h postinfection a robust EV-mediated protein secretion takes place. We identified 2359 human proteins from EVs of IAV-infected macrophages compared with 1448 proteins identified from EVs of control cells. Bioinformatic analysis shows that many proteins involved in translation, like components of spliceosome machinery and the ribosome, are secreted in EVs in response to IAV infection. Our data also shows that EVs derived from IAV-infected macrophages contain fatty acid-binding proteins, antiviral cytokines, copper metabolism Murr-1 domain proteins, and autophagy-related proteins. In addition, our data suggest that secretory autophagy plays a role in activating EV-mediated protein secretion during IAV infection.

Regulation of kynurenine biosynthesis during influenza virus infection.

Influenza A viruses (IAVs) remain serious threats to public health because of the shortage of effective means of control. Developing more effective virus control modalities requires better understanding of virus-host interactions. It has previously been shown that IAV induces the production of kynurenine, which suppresses T-cell responses, enhances pain hypersensitivity and disturbs behaviour in infected animals. However, the regulation of kynurenine biosynthesis during IAV infection remains elusive. Here we showed that IAV infection induced expression of interferons (IFNs), which upregulated production of indoleamine-2,3-dioxygenase (IDO1), which catalysed the kynurenine biosynthesis. Furthermore, IAV attenuated the IDO1 expression and the production of kynurenine through its NS1 protein. Interestingly, inhibition of viral replication prior to IFN induction limited IDO1 expression, while inhibition after did not. Finally, we showed that kynurenine biosynthesis was activated in macrophages in response to other stimuli, such as influenza B virus, herpes simplex virus 1 and 2 as well as bacterial lipopolysaccharides. Thus, the tight regulation of the kynurenine biosynthesis by host cell and, perhaps, pathogen might be a basic signature of a wide range of host-pathogen interactions, which should be taken into account during development of novel antiviral and antibacterial drugs.

Hemin and Cobalt Protoporphyrin Inhibit NLRP3 Inflammasome Activation by Enhancing Autophagy: A Novel Mechanism of Inflammasome Regulation.

Inflammasomes are intracellular protein platforms, which, upon activation, produce the highly proinflammatory cytokines interleukin (IL)-1ß and IL-18. Heme, hemin and their degradation products possess significant immunomodulatory functions. Here, we studied whether hemin regulates inflammasome function in macrophages. Both hemin and its derivative, cobalt protoporphyrin (CoPP), significantly reduced IL-1ß secretion by cultured human primary macrophages, the human monocytic leukemia cell line and also mouse bone marrow-derived and peritoneal macrophages. Intraperitoneal administration of CoPP to mice prior to urate crystal-induced peritonitis alleviated IL-1ß secretion to the peritoneal cavity. In cultured macrophages, hemin and CoPP inhibited NLRP3 inflammasome assembly by reducing the amount of intracellular apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC). The reduction of ASC was associated with enhanced autophagosome formation and autophagic flux. Inhibition of autophagy prevented the CoPP-induced depletion of ASC, implying that the depletion was caused by increased autophagy. Our data indicate that hemin functions as an endogenous negative regulator of the NLRP3 inflammasome. The inhibition is mediated via enhanced autophagy that results in increased degradation of ASC. This regulatory mechanism may provide a novel approach for the treatment of inflammasome-related diseases.

Multi-Omics Studies towards Novel Modulators of Influenza A Virus-Host Interaction.

Human influenza A viruses (IAVs) cause global pandemics and epidemics. These viruses evolve rapidly, making current treatment options ineffective. To identify novel modulators of IAV-host interactions, we re-analyzed our recent transcriptomics, metabolomics, proteomics, phosphoproteomics, and genomics/virtual ligand screening data. We identified 713 potential modulators targeting 199 cellular and two viral proteins. Anti-influenza activity for 48 of them has been reported previously, whereas the antiviral efficacy of the 665 remains unknown. Studying anti-influenza efficacy and immuno/neuro-modulating properties of these compounds and their combinations as well as potential viral and host resistance to them may lead to the discovery of novel modulators of IAV-host interactions, which might be more effective than the currently available anti-influenza therapeutics.

Calpain Activity Is Essential for ATP-Driven Unconventional Vesicle-Mediated Protein Secretion and Inflammasome Activation in Human Macrophages.

Extracellular ATP is an endogenous danger signal that is known to activate inflammatory responses in innate immune cells, including macrophages. Activated macrophages start to secrete proteins to induce an immune response, as well as to recruit other immune cells to the site of infection and tissue damage. In this study, we characterized the secretome (i.e., the global pattern of secreted proteins) of ATP-stimulated human macrophages. We show that ATP stimulation activates robust vesicle-mediated unconventional protein secretion, including exosome release and membrane shedding, from human macrophages. Pathway analysis of the identified secreted proteins showed that calpain-related pathways were overrepresented in the secretome of ATP-stimulated cells. In accordance with this, calpains, which are calcium-dependent nonlysosomal cysteine proteases, were activated upon ATP stimulation through a P2X purinoceptor 7 receptor-dependent pathway. Functional studies demonstrated that calpain activity is essential for the P2X purinoceptor 7 receptor-mediated activation of unconventional protein secretion. Unconventional protein secretion was followed by cell necrosis and NLRP3 inflammasome-mediated secretion of the mature form of the proinflammatory cytokine IL-1ß. Furthermore, ATP-driven NLRP3 inflammasome activation was also dependent on calpain activity. Interestingly, pro-IL-1ß and inflammasome components ASC and caspase-1 were released by ATP-activated macrophages through a vesicle-mediated secretion pathway. In conclusion, to our knowledge, we provide the first global characterization of proteins secreted by ATP-activated human macrophages and show a pivotal role for calpains in the activation of the inflammatory response during ATP exposure.

Influenza virus NS1 protein binds cellular DNA to block transcription of antiviral genes.

Influenza NS1 protein is an important virulence factor that is capable of binding double-stranded (ds) RNA and inhibiting dsRNA-mediated host innate immune responses. Here we show that NS1 can also bind cellular dsDNA. This interaction prevents loading of transcriptional machinery to the DNA, thereby attenuating IAV-mediated expression of antiviral genes. Thus, we identified a previously undescribed strategy, by which RNA virus inhibits cellular transcription to escape antiviral response and secure its replication.

Phosphoproteomics to Characterize Host Response During Influenza A Virus Infection of Human Macrophages.

Influenza A viruses cause infections in the human respiratory tract and give rise to annual seasonal outbreaks, as well as more rarely dreaded pandemics. Influenza A viruses become quickly resistant to the virus-directed antiviral treatments, which are the current main treatment options. A promising alternative approach is to target host cell factors that are exploited by influenza viruses. To this end, we characterized the phosphoproteome of influenza A virus infected primary human macrophages to elucidate the intracellular signaling pathways and critical host factors activated upon influenza infection. We identified 1675 phosphoproteins, 4004 phosphopeptides and 4146 nonredundant phosphosites. The phosphorylation of 1113 proteins (66%) was regulated upon infection, highlighting the importance of such global phosphoproteomic profiling in primary cells. Notably, 285 of the identified phosphorylation sites have not been previously described in publicly available phosphorylation databases, despite many published large-scale phosphoproteome studies using human and mouse cell lines. Systematic bioinformatics analysis of the phosphoproteome data indicated that the phosphorylation of proteins involved in the ubiquitin/proteasome pathway (such as TRIM22 and TRIM25) and antiviral responses (such as MAVS) changed in infected macrophages. Proteins known to play roles in small GTPase-, mitogen-activated protein kinase-, and cyclin-dependent kinase- signaling were also regulated by phosphorylation upon infection. In particular, the influenza infection had a major influence on the phosphorylation profiles of a large number of cyclin-dependent kinase substrates. Functional studies using cyclin-dependent kinase inhibitors showed that the cyclin-dependent kinase activity is required for efficient viral replication and for activation of the host antiviral responses. In addition, we show that cyclin-dependent kinase inhibitors protect IAV-infected mice from death. In conclusion, we provide the first comprehensive phosphoproteome characterization of influenza A virus infection in primary human macrophages, and provide evidence that cyclin-dependent kinases represent potential therapeutic targets for more effective treatment of influenza infections.