A pairwise cytokine code explains the organism-wide response to sepsis.

Sepsis is a systemic response to infection with life-threatening consequences. Our understanding of the molecular and cellular impact of sepsis across organs remains rudimentary. Here, we characterize the pathogenesis of sepsis by measuring dynamic changes in gene expression across organs. To pinpoint molecules controlling organ states in sepsis, we compare the effects of sepsis on organ gene expression to those of 6 singles and 15 pairs of recombinant cytokines. Strikingly, we find that the pairwise effects of tumor necrosis factor plus interleukin (IL)-18, interferon-gamma or IL-1ß suffice to mirror the impact of sepsis across tissues. Mechanistically, we map the cellular effects of sepsis and cytokines by computing changes in the abundance of 195 cell types across 9 organs, which we validate by whole-mouse spatial profiling. Our work decodes the cytokine cacophony in sepsis into a pairwise cytokine message capturing the gene, cell and tissue responses of the host to the disease.

Organism-Wide Analysis of Sepsis Reveals Mechanisms of Systemic Inflammation.

Sepsis is a systemic response to infection with life-threatening consequences. Our understanding of the impact of sepsis across organs of the body is rudimentary. Here, using mouse models of sepsis, we generate a dynamic, organism-wide map of the pathogenesis of the disease, revealing the spatiotemporal patterns of the effects of sepsis across tissues. These data revealed two interorgan mechanisms key in sepsis. First, we discover a simplifying principle in the systemic behavior of the cytokine network during sepsis, whereby a hierarchical cytokine circuit arising from the pairwise effects of TNF plus IL-18, IFN-γ, or IL-1ß explains half of all the cellular effects of sepsis on 195 cell types across 9 organs. Second, we find that the secreted phospholipase PLA2G5 mediates hemolysis in blood, contributing to organ failure during sepsis. These results provide fundamental insights to help build a unifying mechanistic framework for the pathophysiological effects of sepsis on the body.

Nanaomycin E inhibits NLRP3 inflammasome activation by preventing mitochondrial dysfunction.

Nod-like receptor family pyrin domain-containing 3 (NLRP3) is a cytosolic innate immune receptor that senses organelle dysfunction induced by various stimuli, such as infectious, environmental, metabolic and drug stresses. Upon activation, NLRP3 forms an inflammasome with its adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase-1, to trigger the release of inflammatory cytokines. The development of effective anti-inflammatory drugs targeting the NLRP3 inflammasome is in high demand as its aberrant activation often causes inflammatory diseases. Here, we found that nanaomycin A (NNM-A), a quinone-based antibiotic isolated from Streptomyces, effectively inhibited NLRP3 inflammasome-mediated inflammatory responses induced by imidazoquinolines, including imiquimod. Interestingly, its epoxy derivative nanaomycin E (NNM-E) showed a comparable inhibitory effect against the NLRP3 inflammasome-induced release of interleukin (IL)-1ß and IL-18 from macrophages, with a much lower toxicity than NNM-A. NNM-E inhibited ASC oligomerization and caspase-1 cleavage, both of which are hallmarks of NLRP3 inflammasome activation. NNM-E reduced mitochondrial damage and the production of reactive oxygen species, thereby preventing the activation of the NLRP3 inflammasome. NNM-E treatment markedly alleviated psoriasis-like skin inflammation induced by imiquimod. Collectively, NNM-E inhibits NLRP3 inflammasome activation by preventing mitochondrial dysfunction with little toxicity and showed an anti-inflammatory effect in vivo. Thus, NNM-E could be a potential lead compound for developing effective and safe anti-inflammatory agents for the treatment of NLRP3 inflammasome-mediated inflammatory diseases.

Oridonin suppresses particulate-induced NLRP3-independent IL-1α release to prevent crystallopathy in the lung.

The human body is exposed to various particulates of industrial, environmental, or endogenous origin. Invading or intrinsic particulates can induce inflammation by aberrantly activating the immune system, thereby causing crystallopathies. When immune cells such as macrophages phagocytose the particulates, their phagolysosomal membranes undergo mechanical damage, eventually leading to pyroptotic cell death accompanied by the release of inflammatory cytokines, including interleukin (IL)-1α and IL-1ß. The nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is responsible for particulate-induced IL-1ß release and is therefore regarded as a potential therapeutic target for inflammation-mediated crystallopathies. However, IL-1α is released after particulate stimulation in an NLRP3 inflammasome-independent manner and plays a critical role in disease development. Therefore, drugs that exert potent anti-inflammatory effects by comprehensively suppressing particulate-induced responses, including IL-1ß release and IL-1α release, should be developed. Here, we found that oridonin, a diterpenoid isolated from Isodon japonicus HARA, strongly suppressed particulate-induced cell death, accompanied by the release of IL-1α and IL-1ß in mouse and human macrophages. Oridonin reduced particulate-induced phagolysosomal membrane damage in macrophages without affecting phagocytosis of particulates. Furthermore, oridonin treatment markedly suppressed the symptoms of silica particle-induced pneumonia, which was attributed to the release of IL-1α independently of NLRP3. Thus, oridonin is a potential lead compound for developing effective therapeutics for crystallopathies attributed to NLRP3-dependent as well as NLRP3-independent inflammation.

A whole-tissue RNA-seq toolkit for organism-wide studies of gene expression with PME-seq.

The immune system operates at the scale of the whole organism in mammals. We currently lack experimental approaches to systematically track and study organism-wide molecular processes in mice. Here we describe an integrated toolkit for measuring gene expression in whole tissues, 3-prime mRNA extension sequencing, that is applicable to most mouse organs and any mouse model of interest. Further, the methods of RNA-seq described in this protocol are broadly applicable to other sample types beyond whole organs, such as tissue samples or isolated cell populations. We report procedures to collect, store and lyse a dozen organ types using conditions compatible with the extraction of high-quality RNA. In addition, we detail protocols to perform high-throughput and low-cost RNA extraction and sequencing, as well as downstream data analysis. The protocol takes 5 d to process 384 mouse organs from collecting tissues to obtaining raw sequencing data, with additional time required for data analysis and mining. The protocol is accessible to individuals with basic skills in (i) mouse perfusion and dissection for sample collection and (ii) computation using Unix and R for data analysis. Overall, the methods presented here fill a gap in our toolbox for studying organism-wide processes in immunology and physiology.

Autophagy limits activation of the inflammasomes.

Inflammasomes are multiprotein complexes that control the maturation and production of interleukin-1 family members and play crucial roles in host defense against pathogens. However, dysregulated activation of inflammasomes is associated with intense inflammation, leading to the development of inflammatory diseases. Therefore, inflammasomes must be activated at a proper strength to protect against infection and avoid tissue damage. Recent studies have highlighted the cross-talk between inflammasome activation and autophagy, the cellular machinery associated with the degradation of intracellular components and maintenance of cellular homeostasis. Notably, deficiencies in autophagy-related proteins induce the aberrant activation of inflammasomes, causing severe tissue damage. In contrast, autophagy inducers ameliorate symptoms of inflammasome-related diseases. In this review, we discuss recent advances in the involvement of autophagy in regulating inflammasomes activation and in the development of inflammatory diseases.

The RAB2B-GARIL5 Complex Promotes Cytosolic DNA-Induced Innate Immune Responses.

Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that induces the IFN antiviral response. However, the regulatory mechanisms that mediate cGAS-triggered signaling have not been fully explored. Here, we show the involvement of a small GTPase, RAB2B, and its effector protein, Golgi-associated RAB2B interactor-like 5 (GARIL5), in the cGAS-mediated IFN response. RAB2B-deficiency affects the IFN response induced by cytosolic DNA. Consistent with this, RAB2B deficiency enhances replication of vaccinia virus, a DNA virus. After DNA stimulation, RAB2B colocalizes with stimulator of interferon genes (STING), the downstream signal mediator of cGAS, on the Golgi apparatus. The GTP-binding activity of RAB2B is required for its localization on the Golgi apparatus and for recruitment of GARIL5. GARIL5 deficiency also affects the IFN response induced by cytosolic DNA and enhances replication of vaccinia virus. These findings indicate that the RAB2B-GARIL5 complex promotes IFN responses against DNA viruses by regulating the cGAS-STING signaling axis.

Mitochondria-Endoplasmic Reticulum Contact Sites Mediate Innate Immune Responses.

Mitochondria and the endoplasmic reticulum (ER) are fundamental organelles that coordinate high-order cell functions. Mitochondria are centers of energy production, whereas the ER is responsible for folding, transport, and degradation of proteins. In addition to their specific functions, mitochondria and ER actively communicate with each other to promote a variety of cellular events, such as material transfer and signal transduction. Recent studies have shown the critical involvement of these organelles in regulation of the innate immune system, which functions in host defense. The innate immune system utilizes a wide range of germ-line-encoded pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) and induces inflammatory and antiviral responses. Contact sites between mitochondria and the ER function in assembly of the NLR family pyrin domain containing 3 (NLRP3)-inflammasome to promote the inflammatory response. The NLRP3-inflammasome is a protein complex composed of the receptor NLRP3 on the ER side and the adaptor apoptosis-associated speck-like protein containing a CARD on the mitochondrial side; it induces caspase-1-dependent maturation of proinflammatory cytokines such as interleukin (IL)-1ß and IL-18. Furthermore, ER-mitochondria contact sites function in initiation and mediation of signal transduction pathways downstream of intracellular PRRs, such as retinoic acid-inducible gene I-like receptor and cyclic GMP-AMP synthase, to promote the antiviral response. Therefore, ER-mitochondria contact sites, also known as mitochondria-associated membranes, play key roles in regulation of innate immune responses.

Mitochondrial damage elicits a TCDD-inducible poly(ADP-ribose) polymerase-mediated antiviral response.

The innate immune system senses RNA viruses by pattern recognition receptors (PRRs) and protects the host from virus infection. PRRs mediate the production of immune modulatory factors and direct the elimination of RNA viruses. Here, we show a unique PRR that mediates antiviral response. Tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly(ADP ribose) polymerase (TIPARP), a Cysteine3 Histidine (CCCH)-type zinc finger-containing protein, binds to Sindbis virus (SINV) RNA via its zinc finger domain and recruits an exosome to induce viral RNA degradation. TIPARP typically localizes in the nucleus, but it accumulates in the cytoplasm after SINV infection, allowing targeting of cytoplasmic SINV RNA. Redistribution of TIPARP is induced by reactive oxygen species (ROS)-dependent oxidization of the nuclear pore that affects cytoplasmic-nuclear transport. BCL2-associated X protein (BAX) and BCL2 antagonist/killer 1 (BAK1), B-cell leukemia/lymphoma 2 (BCL2) family members, mediate mitochondrial damage to generate ROS after SINV infection. Thus, TIPARP is a viral RNA-sensing PRR that mediates antiviral responses triggered by BAX- and BAK1-dependent mitochondrial damage.

Resveratrol inhibits the acetylated α-tubulin-mediated assembly of the NLRP3-inflammasome.

With its adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), Nod-like receptor family, pyrin domain containing 3 (NLRP3) forms the inflammasome and mediates inflammatory innate immune responses. Development of an anti-inflammatory drug targeting the NLRP3-inflammasome is urgently required because its aberrant activation often causes inflammatory diseases, including gout. We show that resveratrol, a natural polyphenol in grapes and wine, is a safe and effective phytochemical that inhibits NLRP3-inflammasome activation. Resveratrol inhibits the accumulation of acetylated α-tubulin caused by mitochondrial damage in macrophages stimulated with inducers of the NLRP3-inflammasome. Consequently, resveratrol inhibits the acetylated-α-tubulin-mediated spatial arrangement of mitochondria and their subsequent contact with the endoplasmic reticulum (ER), causing insufficient assembly of ASC on the mitochondria and NLRP3 on the ER. These findings indicate that resveratrol targets the generation of an optimal site for the assembly of NLRP3 and ASC, thus inhibiting NLRP3-inflammasome activation. Therefore, resveratrol could be an effective medication for the treatment of NLRP3-related inflammatory diseases.

Role of zinc-finger anti-viral protein in host defense against Sindbis virus.

Accumulating evidence indicates that type I interferon (IFN) mediates the host protective response to RNA viruses. However, the anti-viral effector molecules involved in this response have not been fully identified. Here, we show that zinc-finger anti-viral protein (ZAP), an IFN-inducible gene, plays a critical role in the elimination of Sindbis virus (SINV) in vitro and in vivo. The loss of ZAP greatly enhances the replication of SINV but does not inhibit type I IFN production in primary mouse embryonic fibroblasts (MEFs). ZAP binds and destabilizes SINV RNA, thereby suppressing the replication of SINV. Type I IFN fails to suppress SINV replication in ZAP-deficient MEFs, whereas the ectopic expression of ZAP is sufficient to suppress the replication of SINV in MEFs lacking the expression of type I IFN and the IFN-inducible genes. ZAP-deficient mice are highly susceptible to SINV infection, although they produce sufficient amounts of type I IFN. Therefore, ZAP is an RNA-sensing anti-viral effector molecule that mediates the type-I-IFN-dependent host defense against SINV.

Zinc-finger antiviral protein mediates retinoic acid inducible gene I-like receptor-independent antiviral response to murine leukemia virus.

When host cells are infected by an RNA virus, pattern-recognition receptors (PRRs) recognize the viral RNA and induce the antiviral innate immunity. Toll-like receptor 7 (TLR7) detects the genomic RNA of incoming murine leukemia virus (MLV) in endosomes and mediates the antiviral response. However, the RNA-sensing PRR that recognizes the MLV in the cytosol is not fully understood. Here, we definitively demonstrate that zinc-finger antiviral protein (ZAP) acts as a cytosolic RNA sensor, inducing the degradation of the MLV transcripts by the exosome, an RNA degradation system, on RNA granules. Although the retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 detect various RNA viruses in the cytosol and induce the type I IFN-dependent antiviral response, RLR loss does not alter the replication efficiency of MLV. In sharp contrast, the loss of ZAP greatly enhances the replication efficiency of MLV. ZAP localizes to RNA granules, where the processing-body and stress-granule proteins assemble. ZAP induces the recruitment of the MLV transcripts and exosome components to the RNA granules. The CCCH-type zinc-finger domains of ZAP, which are RNA-binding motifs, mediate its localization to RNA granules and MLV transcripts degradation by the exosome. Although ZAP was known as a regulator of RIG-I signaling in a human cell line, ZAP deficiency does not affect the RIG-I-dependent production of type I IFN in mouse cells. Thus, ZAP is a unique member of the cytosolic RNA-sensing PRR family that targets and eliminates intracellular RNA viruses independently of TLR and RLR family members.

Microtubule-driven spatial arrangement of mitochondria promotes activation of the NLRP3 inflammasome.

NLRP3 forms an inflammasome with its adaptor ASC, and its excessive activation can cause inflammatory diseases. However, little is known about the mechanisms that control assembly of the inflammasome complex. Here we show that microtubules mediated assembly of the NLRP3 inflammasome. Inducers of the NLRP3 inflammasome caused aberrant mitochondrial homeostasis to diminish the concentration of the coenzyme NAD(+), which in turn inactivated the NAD(+)-dependent α-tubulin deacetylase sirtuin 2; this resulted in the accumulation of acetylated α-tubulin. Acetylated α-tubulin mediated the dynein-dependent transport of mitochondria and subsequent apposition of ASC on mitochondria to NLRP3 on the endoplasmic reticulum. Therefore, in addition to direct activation of NLRP3, the creation of optimal sites for signal transduction by microtubules is required for activation of the entire NLRP3 inflammasome.

Neutrophil extracellular traps mediate a host defense response to human immunodeficiency virus-1.

Neutrophils contribute to pathogen clearance by producing neutrophil extracellular traps (NETs), which are genomic DNA-based net-like structures that capture bacteria and fungi. Although NETs also express antiviral factors, such as myeloperoxidase and α-defensin, the involvement of NETs in antiviral responses remains unclear. We show that NETs capture human immunodeficiency virus (HIV)-1 and promote HIV-1 elimination through myeloperoxidase and α-defensin. Neutrophils detect HIV-1 by Toll-like receptors (TLRs) TLR7 and TLR8, which recognize viral nucleic acids. Engagement of TLR7 and TLR8 induces the generation of reactive oxygen species that trigger NET formation, leading to NET-dependent HIV-1 elimination. However, HIV-1 counteracts this response by inducing C-type lectin CD209-dependent production of interleukin (IL)-10 by dendritic cells to inhibit NET formation. IL-10 suppresses the reactive oxygen species-dependent generation of NETs induced upon TLR7 and TLR8 engagement, resulting in disrupted NET-dependent HIV-1 elimination. Therefore, NET formation is an antiviral response that is counteracted by HIV-1.