STING promotes homeostatic maintenance of tissues and confers longevity with aging.

Local immune processes within aging tissues are a significant driver of aging associated dysfunction, but tissue-autonomous pathways and cell types that modulate these responses remain poorly characterized. The cytosolic DNA sensing pathway, acting through cyclic GMP-AMP synthase (cGAS) and Stimulator of Interferon Genes (STING), is broadly expressed in tissues, and is poised to regulate local type I interferon (IFN-I)-dependent and independent inflammatory processes within tissues. Recent studies suggest that the cGAS/STING pathway may drive pathology in various in vitro and in vivo models of accelerated aging. To date, however, the role of the cGAS/STING pathway in physiological aging processes, in the absence of genetic drivers, has remained unexplored. This remains a relevant gap, as STING is ubiquitously expressed, implicated in multitudinous disorders, and loss of function polymorphisms of STING are highly prevalent in the human population (>50%). Here we reveal that, during physiological aging, STING-deficiency leads to a significant shortening of murine lifespan, increased pro-inflammatory serum cytokines and tissue infiltrates, as well as salient changes in histological composition and organization. We note that aging hearts, livers, and kidneys express distinct subsets of inflammatory, interferon-stimulated gene (ISG), and senescence genes, collectively comprising an immune fingerprint for each tissue. These distinctive patterns are largely imprinted by tissue-specific stromal and myeloid cells. Using cellular interaction network analyses, immunofluorescence, and histopathology data, we show that these immune fingerprints shape the tissue architecture and the landscape of cell-cell interactions in aging tissues. These age-associated immune fingerprints are grossly dysregulated with STING-deficiency, with key genes that define aging STING-sufficient tissues greatly diminished in the absence of STING. Changes in immune signatures are concomitant with a restructuring of the stromal and myeloid fractions, whereby cell:cell interactions are grossly altered and resulting in disorganization of tissue architecture in STING-deficient organs. This altered homeostasis in aging STING-deficient tissues is associated with a cross-tissue loss of homeostatic tissue-resident macrophage (TRM) populations in these tissues. Ex vivo analyses reveal that basal STING-signaling limits the susceptibility of TRMs to death-inducing stimuli and determines their in situ localization in tissue niches, thereby promoting tissue homeostasis. Collectively, these data upend the paradigm that cGAS/STING signaling is primarily pathological in aging and instead indicate that basal STING signaling sustains tissue function and supports organismal longevity. Critically, our study urges caution in the indiscriminate targeting of these pathways, which may result in unpredictable and pathological consequences for health during aging.

Neutrophils and macrophages drive TNF-induced lethality via TRIF/CD14-mediated responses.

TNF mediates a variety of biological processes including cellular proliferation, inflammatory responses, and cell death and is therefore associated with numerous pathologies including autoinflammatory diseases and septic shock. The inflammatory and cell death responses to TNF have been studied extensively downstream of TNF-R1 and are believed to rely on the formation of proinflammatory complex I and prodeath complex II, respectively. We recently identified a similar multimeric complex downstream of TLR4, termed the TRIFosome, that regulates inflammation and cell death in response to LPS or Yersinia pseudotuberculosis. We present evidence of a role for the TRIFosome downstream of TNF-R1, independent of TLR3 or TLR4 engagement. Specifically, TNF-induced cell death and inflammation in murine macrophages were driven by the TLR4 adaptor TRIF and the LPS co-receptor CD14, highlighting an important role for these proteins beyond TLR-mediated immune responses. Via immunoprecipitation and visualization of TRIF-specific puncta, we demonstrated TRIF- and CD14-dependent formation of prodeath and proinflammatory complexes in response to TNF. Extending these findings, in a murine TNF-induced sepsis model, TRIF and CD14 deficiency decreased systemic inflammation, reduced organ pathology, and improved survival. The outcome of TRIF activation was cell specific, because TNF-induced lethality was mediated by neutrophils and macrophages responding to TNF in a TRIF-dependent manner. Our findings suggest that in addition to their crucial role in TNF production, myeloid cells are central to TNF toxicity and position TRIF and CD14 as universal components of receptor-mediated immune responses.

Cell death: All roads lead to mitochondria.

Mitochondria are central to apoptosis, an immunologically silent form of cell death. The mitochondrial, or 'intrinsic', apoptotic pathway is activated when the permeabilized mitochondrial membrane of stressed cells releases apoptotic effectors. A new study now characterizes how mitochondria are involved in the switch from pyroptotic to necroptotic cell death.

ZBP1 promotes inflammatory responses downstream of TLR3/TLR4 via timely delivery of RIPK1 to TRIF.

ZBP1 is widely recognized as a mediator of cell death for its role in initiating necroptotic, apoptotic, and pyroptotic cell death pathways in response to diverse pathogenic infection. Herein, we characterize an unanticipated role for ZBP1 in promoting inflammatory responses to bacterial lipopolysaccharide (LPS) or double-stranded RNA (dsRNA). In response to both stimuli, ZBP1 promotes the timely delivery of RIPK1 to the Toll-like receptor (TLR)3/4 adaptor TRIF and M1-ubiquitination of RIPK1, which sustains activation of inflammatory signaling cascades downstream of RIPK1. Strikingly, ZBP1-mediated regulation of these pathways is important in vivo, as Zbp1−/− mice exhibited resistance to LPS-induced septic shock, revealed by prolonged survival and delayed onset of hypothermia due to decreased inflammatory responses and subsequent cell death. Further findings revealed that ZBP1 promotes sustained inflammatory responses by mediating the kinetics of proinflammatory "TRIFosome" complex formation, thus having a profound impact downstream of TLR activation. Given the well-characterized role of ZBP1 as a viral sensor, our results exemplify previously unappreciated crosstalk between the pathways that regulate host responses to bacteria and viruses, with ZBP1 acting as a crucial bridge between the two.

Replicating Cortical Signatures May Open the Possibility for "Transplanting" Brain States via Brain Entrainment.

Brain states, which correlate with specific motor, cognitive, and emotional states, may be monitored with noninvasive techniques such as electroencephalography (EEG) and magnetoencephalography (MEG) that measure macroscopic cortical activity manifested as oscillatory network dynamics. These rhythmic cortical signatures provide insight into the neuronal activity used to identify pathological cortical function in numerous neurological and psychiatric conditions. Sensory and transcranial stimulation, entraining the brain with specific brain rhythms, can effectively induce desired brain states (such as state of sleep or state of attention) correlated with such cortical rhythms. Because brain states have distinct neural correlates, it may be possible to induce a desired brain state by replicating these neural correlates through stimulation. To do so, we propose recording brain waves from a "donor" in a particular brain state using EEG/MEG to extract cortical signatures of the brain state. These cortical signatures would then be inverted and used to entrain the brain of a "recipient" via sensory or transcranial stimulation. We propose that brain states may thus be transferred between people by acquiring an associated cortical signature from a donor, which, following processing, may be applied to a recipient through sensory or transcranial stimulation. This technique may provide a novel and effective neuromodulation approach to the noninvasive, non-pharmacological treatment of a variety of psychiatric and neurological disorders for which current treatments are mostly limited to pharmacotherapeutic interventions.

Endothelial STING controls T cell transmigration in an IFNI-dependent manner.

The stimulator of IFN genes (STING) protein senses cyclic dinucleotides released in response to double-stranded DNA and functions as an adaptor molecule for type I IFN (IFNI) signaling by activating IFNI-stimulated genes (ISG). We found impaired T cell infiltration into the peritoneum in response to TNF-α in global and EC-specific STING-/- mice and discovered that T cell transendothelial migration (TEM) across mouse and human endothelial cells (EC) deficient in STING was strikingly reduced compared with control EC, whereas T cell adhesion was not impaired. STING-/- T cells showed no defect in TEM or adhesion to EC, or immobilized endothelial cell-expressed molecules ICAM1 and VCAM1, compared with WT T cells. Mechanistically, CXCL10, an ISG and a chemoattractant for T cells, was dramatically reduced in TNF-α-stimulated STING-/- EC, and genetic loss or pharmacologic antagonisms of IFNI receptor (IFNAR) pathway reduced T cell TEM. Our data demonstrate a central role for EC-STING during T cell TEM that is dependent on the ISG CXCL10 and on IFNI/IFNAR signaling.

Immunoprecipitation Strategies to Isolate RIPK1/RIPK3 Complexes in Mouse Macrophages.

A large protein complex, containing RIPK1, RIPK3, and caspase-8 and known as Complex II, has emerged as one of the key mediators of cell death downstream from a range of innate immune triggers. This regulatory mechanism plays a prominent role in macrophages, where Complex II has been linked to apoptosis, pyroptosis, and necroptosis as well as the enhancement of inflammatory gene expression. Although core components of this complex are fairly well understood, more subtle proteomic changes that determine the direction of a response once the complex is assembled remain much less clear. In addition, Complex II components undergo a wealth of post-translational changes that modify the functions of the complex components. This necessitates development of robust and efficient methods of isolating Complex II for further interrogation of its composition and the post-translational modifications of its components. This article describes several methods that we have developed for Complex II isolation, which can be used to obtain complementary information about this signaling mechanism. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Isolation of Complex II in necroptotic and pyroptotic macrophages using FADD immunoprecipitation Basic Protocol 2: Isolation of the complexes formed by the conditionally expressed 3XFLAG-RIPK1 protein Alternate Protocol: Alternative methods of immunoprecipitation of RIPK1 and other Complex-II-related factors Support Protocol: Generation of stable macrophage cell lines using lentiviral expression Basic Protocol 3: Use of proximity labeling to identify necrosome components in the detergent-insoluble fraction of the cell lysates.

ZBP1 promotes LPS-induced cell death and IL-1ß release via RHIM-mediated interactions with RIPK1.

Inflammation and cell death are closely linked arms of the host immune response to infection, which when carefully balanced ensure host survival. One example of this balance is the tightly regulated transition from TNFR1-associated pro-inflammatory complex I to pro-death complex II. By contrast, here we show that a TRIF-dependent complex containing FADD, RIPK1 and caspase-8 (that we have termed the TRIFosome) mediates cell death in response to Yersinia pseudotuberculosis and LPS. Furthermore, we show that constitutive binding between ZBP1 and RIPK1 is essential for the initiation of TRIFosome interactions, caspase-8-mediated cell death and inflammasome activation, thus positioning ZBP1 as an effector of cell death in the context of bacterial blockade of pro-inflammatory signaling. Additionally, our findings offer an alternative to the TNFR1-dependent model of complex II assembly, by demonstrating pro-death complex formation reliant on TRIF signaling.

Flipping the Switch from Inflammation to Cell Death.

Multiple research groups have demonstrated that caspase-8 (CASP8)-mediated gasdermin D (GSDMD) cleavage drives pyroptotic cell death. Here, we discuss a novel role for the enzymatically inactive homolog of CASP8, the long isoform of cellular FLICE-like inhibitory protein (cFLIPL), in the regulation of this process. Specifically, cFLIP-deficiency provides a model in which to study the mechanisms regulating CASP8-mediated activation of cell death and inflammatory signaling.

cFLIPL protects macrophages from LPS-induced pyroptosis via inhibition of complex II formation.

Cell death and inflammation are interdependent host responses to infection. During pyroptotic cell death, interleukin-1ß (IL-1ß) release occurs through caspase-1 and caspase-11-mediated gasdermin D pore formation. In vivo, responses to lipopolysaccharide (LPS) result in IL-1ß secretion. In vitro, however, murine macrophages require a second "danger signal" for the inflammasome-driven maturation of IL-1ß. Recent reports have shown caspase-8-mediated pyroptosis in LPS-activated macrophages but have provided conflicting evidence regarding the release of IL-1ß under these conditions. Here, to further characterize the mechanism of LPS-induced secretion in vitro, we reveal an important role for cellular FLICE-like inhibitory protein (cFLIP) in the regulation of the inflammatory response. Specifically, we show that deficiency of the long isoform cFLIPL promotes complex II formation, driving pyroptosis, and the secretion of IL-1ß in response to LPS alone.

Constitutive Interferon Attenuates RIPK1/3-Mediated Cytokine Translation.

Receptor-interacting protein kinase 1 (RIPK1) and 3 (RIPK3) are well known for their capacity to drive necroptosis via mixed-lineage kinase-like domain (MLKL). Recently, RIPK1/3 kinase activity has been shown to drive inflammation via activation of MAPK signaling. However, the regulatory mechanisms underlying this kinase-dependent cytokine production remain poorly understood. In the present study, we establish that the kinase activity of RIPK1/3 regulates cytokine translation in mouse and human macrophages. Furthermore, we show that this inflammatory response is downregulated by type I interferon (IFN) signaling, independent of type I IFN-promoted cell death. Specifically, low-level constitutive IFN signaling attenuates RIPK-driven activation of cap-dependent translation initiation pathway components AKT, mTORC1, 4E-BP and eIF4E, while promoting RIPK-dependent cell death. Altogether, these data characterize constitutive IFN signaling as a regulator of RIPK-dependent inflammation and establish cap-dependent translation as a crucial checkpoint in the regulation of cytokine production.

Correction: Nikitin, D., et al. Retroelement-Linked Transcription Factor Binding Patterns Point to Quickly Developing Molecular Pathways in Human Evolution. Cells 2019, 8, 130.

In the article 'Retroelement-Linked Transcription Factor Binding Patterns Point to Quickly Developing Molecular Pathways in Human Evolution,' a number of transcription factor binding sites (TFBS) mapped on all retroelement classes were incorrectly calculated as sum of TFBS numbers separately mapped on LINEs, SINEs and LTR retrotransposons/endogenous retroviruses (LR/ERVs) [...].

Retroelement-Linked Transcription Factor Binding Patterns Point to Quickly Developing Molecular Pathways in Human Evolution.

BACKGROUND: Retroelements (REs) are transposable elements occupying ~40% of the human genome that can regulate genes by providing transcription factor binding sites (TFBS). RE-linked TFBS profile can serve as a marker of gene transcriptional regulation evolution. This approach allows for interrogating the regulatory evolution of organisms with RE-rich genomes. We aimed to characterize the evolution of transcriptional regulation for human genes and molecular pathways using RE-linked TFBS accumulation as a metric. Methods: We characterized human genes and molecular pathways either enriched or deficient in RE-linked TFBS regulation. We used ENCODE database with mapped TFBS for 563 transcription factors in 13 human cell lines. For 24,389 genes and 3124 molecular pathways, we calculated the score of RE-linked TFBS regulation reflecting the regulatory evolution rate at the level of individual genes and molecular pathways. Results: The major groups enriched by RE regulation deal with gene regulation by microRNAs, olfaction, color vision, fertilization, cellular immune response, and amino acids and fatty acids metabolism and detoxication. The deficient groups were involved in translation, RNA transcription and processing, chromatin organization, and molecular signaling. Conclusion: We identified genes and molecular processes that have characteristics of especially high or low evolutionary rates at the level of RE-linked TFBS regulation in human lineage.

Constitutive interferon signaling maintains critical threshold of MLKL expression to license necroptosis.

Interferons (IFNs) are critical determinants in immune-competence and autoimmunity, and are endogenously regulated by a low-level constitutive feedback loop. However, little is known about the functions and origins of constitutive IFN. Recently, lipopolysaccharide (LPS)-induced IFN was implicated as a driver of necroptosis, a necrotic form of cell death downstream of receptor-interacting protein (RIP) kinase activation and executed by mixed lineage kinase like-domain (MLKL) protein. We found that the pre-established IFN status of the cell, instead of LPS-induced IFN, is critical for the early initiation of necroptosis in macrophages. This pre-established IFN signature stems from cytosolic DNA sensing via cGAS/STING, and maintains the expression of MLKL and one or more unknown effectors above a critical threshold to allow for MLKL oligomerization and cell death. Finally, we found that elevated IFN-signaling in systemic lupus erythematosus (SLE) augments necroptosis, providing a link between pathological IFN and tissue damage during autoimmunity.

Caspase-8 induces cleavage of gasdermin D to elicit pyroptosis during Yersinia infection.

Cell death and inflammation are intimately linked during Yersinia infection. Pathogenic Yersinia inhibits the MAP kinase TGFß-activated kinase 1 (TAK1) via the effector YopJ, thereby silencing cytokine expression while activating caspase-8-mediated cell death. Here, using Yersinia pseudotuberculosis in corroboration with costimulation of lipopolysaccharide and (5Z)-7-Oxozeaenol, a small-molecule inhibitor of TAK1, we show that caspase-8 activation during TAK1 inhibition results in cleavage of both gasdermin D (GSDMD) and gasdermin E (GSDME) in murine macrophages, resulting in pyroptosis. Loss of GsdmD delays membrane rupture, reverting the cell-death morphology to apoptosis. We found that the Yersinia-driven IL-1 response arises from asynchrony of macrophage death during bulk infections in which two cellular populations are required to provide signal 1 and signal 2 for IL-1α/ß release. Furthermore, we found that human macrophages are resistant to YopJ-mediated pyroptosis, with dampened IL-1ß production. Our results uncover a form of caspase-8-mediated pyroptosis and suggest a hypothesis for the increased sensitivity of humans to Yersinia infection compared with the rodent reservoir.

Host-Intrinsic Interferon Status in Infection and Immunity.

Most genetic ablations of interferon (IFN) signaling abolish both the experimentally induced IFN response and constitutive IFN, whose effects are well established in autoimmunity but understudied during infection. In host-pathogen interactions, most IFN-mediated responses are attributed to infection-driven IFN. However, IFNs confer their activity by regulating networks of interferon-stimulated genes (ISGs), a process that requires de novo transcription and translation of both IFN and downstream ISGs through feedback of IFN receptor signaling. Due to the temporal requirement for IFN activity, many rapid antimicrobial responses may instead result from pre-established IFN signature stemming from host-intrinsic processes. Addressing the permeating effects of constitutive IFN is therefore needed to accurately describe immunity as host intrinsic or pathogen induced.

Wild-derived mice: from genetic diversity to variation in immune responses.

Classical inbred mouse strains have historically been instrumental in mapping immunological traits. However, most of the classical strains originate from a relatively limited number of founder animals, largely within the Mus musculus domesticus subspecies. Therefore, their genetic diversity is ultimately limited. For this reason, it is not feasible to use these mice for exhaustive interrogation of immune signaling pathways. In order to investigate networks through forward genetic analysis, larger genetic diversity is required than is introduced under laboratory conditions. Recently, inbred strains from other mouse subspecies were established such as Mus musculus castaneus and Mus musculus musculus, which diverged from a shared common ancestor with Mus musculus domesticus more than one million years ago. A direct genomic comparison clearly demonstrates the evolutionary divergence that has occurred between wild-derived mice and the classical inbred strains. When compared to classical inbred strains, wild-derived mice exhibit polymorphisms every 100-200 base pairs. Studying the molecular basis of these traits provides us with insight into how the immune system can evolve regulatory features to accommodate environment-specific constraints. Because most wild-derived strains are able to breed with classical inbred mice, they represent a rich source of evolutionarily significant diversity for forward genetic studies. These organisms are an emerging, though still largely unexplored, model for the identification and study of novel immunological genes.

Constitutive Interferon Maintains GBP Expression Required for Release of Bacterial Components Upstream of Pyroptosis and Anti-DNA Responses.

Legionella pneumophila elicits caspase-11-driven macrophage pyroptosis through guanylate-binding proteins (GBPs) encoded on chromosome 3. It has been proposed that microbe-driven IFN upregulates GBPs to facilitate pathogen vacuole rupture and bacteriolysis preceding caspase-11 activation. We show here that macrophage death occurred independently of microbial-induced IFN signaling and that GBPs are dispensable for pathogen vacuole rupture. Instead, the host-intrinsic IFN status sustained sufficient GBP expression levels to drive caspase-1 and caspase-11 activation in response to cytosol-exposed bacteria. In addition, endogenous GBP levels were sufficient for the release of DNA from cytosol-exposed bacteria, preceding the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway for Ifnb induction. Mice deficient for chromosome 3 GBPs were unable to mount a rapid IL-1/chemokine (C-X-C motif) ligand 1 (CXCL1) response during Legionella-induced pneumonia, with defective bacterial clearance. Our results show that rapid GBP activity is controlled by host-intrinsic cytokine signaling and that GBP activities precede immune amplification responses, including IFN induction, inflammasome activation, and cell death.

Profiling of Human Molecular Pathways Affected by Retrotransposons at the Level of Regulation by Transcription Factor Proteins.

Endogenous retroviruses and retrotransposons also termed retroelements (REs) are mobile genetic elements that were active until recently in human genome evolution. REs regulate gene expression by actively reshaping chromatin structure or by directly providing transcription factor binding sites (TFBSs). We aimed to identify molecular processes most deeply impacted by the REs in human cells at the level of TFBS regulation. By using ENCODE data, we identified ~2 million TFBS overlapping with putatively regulation-competent human REs located in 5-kb gene promoter neighborhood (~17% of all TFBS in promoter neighborhoods; ~9% of all RE-linked TFBS). Most of REs hosting TFBS were highly diverged repeats, and for the evolutionary young (0-8% diverged) elements we identified only ~7% of all RE-linked TFBS. The gene-specific distributions of RE-linked TFBS generally correlated with the distributions for all TFBS. However, several groups of molecular processes were highly enriched in the RE-linked TFBS regulation. They were strongly connected with the immunity and response to pathogens, with the negative regulation of gene transcription, ubiquitination, and protein degradation, extracellular matrix organization, regulation of STAT signaling, fatty acids metabolism, regulation of GTPase activity, protein targeting to Golgi, regulation of cell division and differentiation, development and functioning of perception organs and reproductive system. By contrast, the processes most weakly affected by the REs were linked with the conservative aspects of embryo development. We also identified differences in the regulation features by the younger and older fractions of the REs. The regulation by the older fraction of the REs was linked mainly with the immunity, cell adhesion, cAMP, IGF1R, Notch, Wnt, and integrin signaling, neuronal development, chondroitin sulfate and heparin metabolism, and endocytosis. The younger REs regulate other aspects of immunity, cell cycle progression and apoptosis, PDGF, TGF beta, EGFR, and p38 signaling, transcriptional repression, structure of nuclear lumen, catabolism of phospholipids, and heterocyclic molecules, insulin and AMPK signaling, retrograde Golgi-ER transport, and estrogen signaling. The immunity-linked pathways were highly represented in both categories, but their functional roles were different and did not overlap. Our results point to the most quickly evolving molecular pathways in the recent and ancient evolution of human genome.