Critical Role of Aquaporins in Interleukin 1β (IL-1β)-induced Inflammation

Rapid changes in cell volume characterize macrophage activation, but the role of water channels in inflammation remains unclear. We show here that, in vitro, aquaporin (AQP) blockade or deficiency results in reduced IL-1β release by macrophages activated with a variety of NLRP3 activators. Inhibition of AQP specifically during the regulatory volume decrease process is sufficient to limit IL-1β release by macrophages through the NLRP3 inflammasome axis. The immune-related activity of AQP was confirmed in vivo in a model of acute lung inflammation induced by crystals. AQP1 deficiency is associated with a marked reduction of both lung IL-1β release and neutrophilic inflammation. We conclude that AQP-mediated water transport in macrophages constitutes a general danger signal required for NLRP3-related inflammation. Our findings reveal a new function of AQP in the inflammatory process and suggest a novel therapeutic target for anti-inflammatory therapy.     

Attenuation of cGAS‐STING signaling is mediated by a p62/SQSTM1‐dependent autophagy pathway activated by TBK1

Negative regulation of immune pathways is essential to achieve resolution of immune responses and to avoid excess inflammation. DNA stimulates type I IFN expression through the DNA sensor cGAS, the second messenger cGAMP, and the adaptor molecule STING. Here, we report that STING degradation following activation of the pathway occurs through autophagy and is mediated by p62/SQSTM1, which is phosphorylated by TBK1 to direct ubiquitinated STING to autophagosomes. Degradation of STING was impaired in p62‐deficient cells, which responded with elevated IFN production to foreign DNA and DNA pathogens. In the absence of p62, STING failed to traffic to autophagy‐associated vesicles. Thus, DNA sensing induces the cGAS‐STING pathway to activate TBK1, which phosphorylates IRF3 to induce IFN expression, but also phosphorylates p62 to stimulate STING degradation and attenuation of the response.     

Dysfunctional telomeres through mitostress‐induced cGAS/STING activation to aggravate immune senescence and viral pneumonia

Disproportionately high incidence and mortality of respiratory infection such as influenza A virus (IAV) and SARS‐CoV‐2 have been evidenced in the elderly, but the role and the mechanism of age‐associated immune deregulation in disease exacerbation are not well defined. Using a late generation of mice deficient in telomerase RNA (Terc^−/−), we herein demonstrated that aged mice were exquisitely susceptible to respiratory viral infection, with excessive inflammation and increased mortality. Furthermore, we identified the cGAS/STING pathway, which was essentially induced by the leaked mitochondrial DNA, as a biologically relevant mechanism contributing to exaggerated inflammation in Terc^−/− mice following viral infection. Innate immune cells, mainly, macrophages with shortened telomeres, exhibited hallmarks of cellular senescence, mitochondrial distress, and aberrant activation of STING and NLRP3 inflammasome pathways, which predisposed mice to severe viral pneumonia during commonly mild infections. Application of STING inhibitor and, more importantly, senolytic agent, reduced the burden of stressed macrophages, improved mitochondrial integrity, and suppressed STING activation, thereby conferring the protection for Terc^−/− mice against respiratory infection. Together, the findings expand our understanding of innate immune senescence and reveal the potential of the senolytics as a promising treatment to alleviate the symptom of viral pneumonia, particularly for the older population.     

Vesicular trafficking permits evasion of cGAS/STING surveillance during initial human papillomavirus infection

Oncogenic human papillomaviruses (HPVs) replicate in differentiating epithelium, causing 5% of cancers worldwide. Like most other DNA viruses, HPV infection initiates after trafficking viral genome (vDNA) to host cell nuclei. Cells possess innate surveillance pathways to detect microbial components or physiological stresses often associated with microbial infections. One of these pathways, cGAS/STING, induces IRF3-dependent antiviral interferon (IFN) responses upon detection of cytosolic DNA. Virion-associated vDNA can activate cGAS/STING during initial viral entry and uncoating/trafficking, and thus cGAS/STING is an obstacle to many DNA viruses. HPV has a unique vesicular trafficking pathway compared to many other DNA viruses. As the capsid uncoats within acidic endosomal compartments, minor capsid protein L2 protrudes across vesicular membranes to facilitate transport of vDNA to the Golgi. L2/vDNA resides within the Golgi lumen until G2/M, whereupon vesicular L2/vDNA traffics along spindle microtubules, tethering to chromosomes to access daughter cell nuclei. L2/vDNA-containing vesicles likely remain intact until G1, following nuclear envelope reformation. We hypothesize that this unique vesicular trafficking protects HPV from cGAS/STING surveillance. Here, we investigate cGAS/STING responses to HPV infection. DNA transfection resulted in acute cGAS/STING activation and downstream IFN responses. In contrast, HPV infection elicited minimal cGAS/STING and IFN responses. To determine the role of vesicular trafficking in cGAS/STING evasion, we forced premature viral penetration of vesicular membranes with membrane-perturbing cationic lipids. Such treatment renders a non-infectious trafficking-defective mutant HPV infectious, yet susceptible to cGAS/STING detection. Overall, HPV evades cGAS/STING by its unique subcellular trafficking, a property that may contribute to establishment of infection.     

Mitochondrial inner membrane permeabilisation enables mtDNA release during apoptosis

During apoptosis, pro‐apoptotic BAX and BAK are activated, causing mitochondrial outer membrane permeabilisation (MOMP), caspase activation and cell death. However, even in the absence of caspase activity, cells usually die following MOMP. Such caspase‐independent cell death is accompanied by inflammation that requires mitochondrial DNA (mtDNA) activation of cGAS‐STING signalling. Because the mitochondrial inner membrane is thought to remain intact during apoptosis, we sought to address how matrix mtDNA could activate the cytosolic cGAS‐STING signalling pathway. Using super‐resolution imaging, we show that mtDNA is efficiently released from mitochondria following MOMP. In a temporal manner, we find that following MOMP, BAX/BAK‐mediated mitochondrial outer membrane pores gradually widen. This allows extrusion of the mitochondrial inner membrane into the cytosol whereupon it permeablises allowing mtDNA release. Our data demonstrate that mitochondrial inner membrane permeabilisation (MIMP) can occur during cell death following BAX/BAK‐dependent MOMP. Importantly, by enabling the cytosolic release of mtDNA, inner membrane permeabilisation underpins the immunogenic effects of caspase‐independent cell death.     

Human ZBP1 induces cell death‐independent inflammatory signaling via RIPK3 and RIPK1

ZBP1 is an interferon‐induced cytosolic nucleic acid sensor that facilitates antiviral responses via RIPK3. Although ZBP1‐mediated programmed cell death is widely described, whether and how it promotes inflammatory signaling is unclear. Here, we report a ZBP1‐induced inflammatory signaling pathway mediated by K63‐ and M1‐linked ubiquitin chains, which depends on RIPK1 and RIPK3 as scaffolds independently of cell death. In human HT29 cells, ZBP1 associated with RIPK1 and RIPK3 as well as ubiquitin ligases cIAP1 and LUBAC. ZBP1‐induced K63‐ and M1‐linked ubiquitination of RIPK1 and ZBP1 to promote TAK1‐ and IKK‐mediated inflammatory signaling and cytokine production. Inhibition of caspase activity suppressed ZBP1‐induced cell death but enhanced cytokine production in a RIPK1‐ and RIPK3 kinase activity‐dependent manner. Lastly, we provide evidence that ZBP1 signaling contributes to SARS‐CoV‐2‐induced cytokine production. Taken together, we describe a ZBP1‐RIPK3‐RIPK1‐mediated inflammatory signaling pathway relayed by the scaffolding role of RIPKs and regulated by caspases, which may induce inflammation when ZBP1 is activated below the threshold needed to trigger a cell death response.     

Cancer cell-specific cGAS/STING Signaling pathway in the era of advancing cancer cell biology

Pattern-recognition receptors (PRRs) are critical to recognizing endogenous and exogenous threats to mount a protective proinflammatory innate immune response. PRRs may be located on the outer cell membrane, cytosol, and nucleus. The cGAS/STING signaling pathway is a cytosolic PRR system. Notably, cGAS is also present in the nucleus. The cGAS-mediated recognition of cytosolic dsDNA and its cleavage into cGAMP activates STING. Furthermore, STING activation through its downstream signaling triggers different interferon-stimulating genes (ISGs), initiating the release of type 1 interferons (IFNs) and NF-κB-mediated release of proinflammatory cytokines and molecules. Activating cGAS/STING generates type 1 IFN, which may prevent cellular transformation and cancer development, growth, and metastasis. The current article delineates the impact of the cancer cell-specific cGAS/STING signaling pathway alteration in tumors and its impact on tumor growth and metastasis. This article further discusses different approaches to specifically target cGAS/STING signaling in cancer cells to inhibit tumor growth and metastasis in conjunction with existing anticancer therapies.     

Human papillomaviruses sensitize cells to DNA damage induced apoptosis by targeting the innate immune sensor cGAS

The cyclic GMP-AMP synthase (cGAS) is a critical regulator of the innate immune response acting as a sensor of double-strand DNAs from pathogens or damaged host DNA. Upon activation, cGAS signals through the STING/TBK1/IRF3 pathway to induce interferon expression. Double stranded DNA viruses target the cGAS pathway to facilitate infection. In HPV positive cells that stably maintain viral episomes, the levels of cGAS were found to be significantly increased over those seen in normal human keratinocytes. Furthermore the downstream effectors of the cGAS pathway, STING and IRF3, were fully active in response to signaling from the secondary messenger cGAMP or poly (dA:dT). In HPV positive cells cGAS was detected in both cytoplasmic puncta as well as in DNA damage induced micronuclei. E6 was responsible for increased levels of cGAS that was dependent on inhibition of p53. CRISPR-Cas9 mediated knockout of cGAS prevented activation of STING and IRF3 but had a minimal effect on viral replication. A primary function of cGAS in HPV positive cells was in response to treatment with etoposide or cisplatin which lead to increased levels of H2AX phosphorylation and activation of caspase 3/7 cleavage while having only a minimal effect on activation of homologous recombination repair factors ATM, ATR or CHK2. In HPV positive cells cGAS was found to regulate the levels of the phosphorylated non-homologous end-joining kinase, DNA-PK, which may contribute to H2AX phosphorylation along with other factors. Importantly cGAS was also responsible for increased levels of DNA breaks along with enhanced apoptosis in HPV positive cells but not in HFKs. This study identifies an important and novel role for cGAS in mediating the response of HPV positive cells to chemotherapeutic drugs.     

Olive phenols preserve lamin B1 expression reducing cGAS/STING/NFκB‐mediated SASP in ionizing radiation‐induced senescence

Senescence occurs upon critical telomere shortening, or following DNA damage, oncogenic activation, hypoxia and oxidative stress, overall referred to stress‐induced premature senescence (SIPS). In response to DNA damage, senescent cells release cytoplasmic chromatin fragments (CCFs), and express an altered secretome, the senescence‐associated secretory phenotype (SASP), which contributes to generate a pro‐inflammatory and pro‐tumoral extracellular milieu. Polyphenols have gained significant attention owing to their anti‐inflammatory and anti‐tumour activities. Here, we studied the effect of oleuropein aglycone (OLE) and hydroxytyrosol (HT) on DNA damage, CCF appearance and SASP in a model of irradiation‐induced senescence. Neonatal human dermal fibroblasts (NHDFs) were γ‐irradiated and incubated with OLE, 5 µM and HT, 1 µM. Cell growth and senescence‐associated (SA)‐β‐Gal‐staining were used as senescence markers. DNA damage was evaluated by Comet assay, lamin B1 expression, release of CCFs, cyclic GMP‐AMP Synthase (cGAS) activation. IL‐6, IL‐8, MCP‐1 and RANTES were measured by ELISA assay. Our results showed that OLE and HT exerted a protective effect on 8 Gy irradiation‐induced senescence, preserving lamin B1 expression and reducing cGAS/STING/NFκB‐mediated SASP. The ability of OLE and HT to mitigate DNA damage, senescence status and the related SASP in normal cells can be exploited to improve the efficacy and safety of cancer radiotherapy.     

Aging aggravated liver ischemia and reperfusion injury by promoting STING‐mediated NLRP3 activation in macrophages

Although aggravated liver injury has been reported in aged livers post‐ischemia and reperfusion (IR), the underlying mechanism of innate immune activation of aged macrophages is not well understood. Here, we investigated whether and how Stimulator of interferon genes (STING) signaling regulated macrophage proinflammatory activation and liver IR injury. Mice were subjected to hepatic IR in vivo. Macrophages isolated from IR‐stressed livers and bone marrow‐derived macrophages (BMDMs) from young and aged mice were used for in vitro studies. Enhanced nucleotide‐binding domain and leucine‐rich repeat containing protein 3 (NLRP3) activation was found in both livers and macrophages of aged mice post‐IR. NLRP3 knockdown in macrophages inhibited intrahepatic inflammation and liver injury in both young and aged mice. Interestingly, enhanced activation of the STING/ TANK‐binding kinase 1 (TBK1) signaling pathway was observed in aged macrophages post‐IR and mitochondria DNA (mtDNA) stimulation. STING suppression blocked over‐activation of NLRP3 signaling and excessive secretion of proinflammatory cytokines/chemokines in the mtDNA‐stimulated BMDMs from aged mice. More importantly, STING knockdown in macrophages abrogated the detrimental role of aging in aggravating liver IR injury and intrahepatic inflammation. Finally, peripheral blood from the recipients undergoing liver transplantation was collected and analyzed. The results showed that the elderly recipients had much higher levels of TNF‐α, IL‐6, IL‐1β, and IL‐18 post‐transplantation, indicating increased NLRP3 activation in lR‐stressed livers of elderly recipients. In summary, our study demonstrated that the STING‐NLRP3 axis was critical for the proinflammatory response of aged macrophages and would be a novel therapeutic target to reduce IR injury in elderly patients.     

Varicella‐Zoster virus ORF9 is an antagonist of the DNA sensor cGAS

Varicella‐Zoster virus (VZV) causes chickenpox and shingles. Although the infection is associated with severe morbidity in some individuals, molecular mechanisms that determine innate immune responses remain poorly defined. We found that the cGAS/STING DNA sensing pathway was required for type I interferon (IFN) induction during VZV infection and that recognition of VZV by cGAS restricted its replication. Screening of a VZV ORF expression library identified the essential VZV tegument protein ORF9 as a cGAS antagonist. Ectopically or virally expressed ORF9 bound to endogenous cGAS leading to reduced type I IFN responses to transfected DNA. Confocal microscopy revealed co‐localisation of cGAS and ORF9. ORF9 and cGAS also interacted directly in a cell‐free system and phase‐separated together with DNA. Furthermore, ORF9 inhibited cGAMP production by cGAS. Taken together, these results reveal the importance of the cGAS/STING DNA sensing pathway for VZV recognition and identify a VZV immune antagonist that partially but directly interferes with DNA sensing via cGAS.     

Endogenous tRNA-derived small RNA (tRF3-Thr-AGT) inhibits ZBP1/NLRP3 pathway-mediated cell pyroptosis to attenuate acute pancreatitis (AP)

Endogenous transfer RNA-derived small RNAs (tsRNAs) are newly identified RNAs that are closely associated with the pathogenesis of multiple diseases, but the involvement of tsRNAs in regulating acute pancreatitis (AP) development has not been reported. In this study, we screened out a novel tsRNA, tRF3-Thr-AGT, that was aberrantly downregulated in the acinar cell line AR42J treated with sodium taurocholate (STC) and the pancreatic tissues of STC-induced AP rat models. In addition, STC treatment suppressed cell viability, induced pyroptotic cell death and cellular inflammation in AP models in vitro and in vivo. Overexpression of tRF3-Thr-AGT partially reversed STC-induced detrimental effects on the AR42J cells. Next, Z-DNA-binding protein 1 (ZBP1) was identified as the downstream target of tRF3-Thr-AGT. Interestingly, upregulation of tRF3-Thr-AGT suppressed NOD-like receptor protein 3 (NLRP3)-mediated pyroptotic cell death in STC-treated AR42J cells via degrading ZBP1. Moreover, the effects of tRF3-Thr-AGT overexpression on cell viability and inflammation in AR42J cells were abrogated by upregulating ZBP1 and NLRP3. Collectively, our data indicated that tRF3-Thr-AGT suppressed ZBP1 expressions to restrain NLRP3-mediated pyroptotic cell death and inflammation in AP models. This study, for the first time, identified the role and potential underlying mechanisms by which tRF3-Thr-AGT regulated AP pathogenesis.     

Emerging insights into molecular mechanisms underlying pyroptosis and functions of inflammasomes in diseases

Pyroptosis is a form of necrotic and inflammatory programmed cell death, which could be characterized by cell swelling, pore formation on plasma membranes, and release of proinflammatory cytokines (IL-1β and IL-18). The process of pyroptosis presents as dual effects: protecting multicellular organisms from microbial infection and endogenous dangers; leading to pathological inflammation if overactivated. Two pathways have been found to trigger pyroptosis: caspase-1 mediated inflammasome pathway with the involvement of NLRP1-, NLRP3-, NLRC4-, AIM2-, pyrin-inflammasome (canonical inflammasome pathway) and caspase-4/5/11-mediated inflammasome pathway (noncanonical inflammasome pathway). Gasdermin D (GSDMD) has been proved to be a substrate of inflammatory caspases (caspase-1/4/5/11), and the cleaved N-terminal domain of GSDMD oligomerizes to form cytotoxic pores on the plasma membrane. Here, we mainly reviewed the up to date mechanisms of pyroptosis, and began with the inflammasomes as the activator of caspase-1/caspase-11, 4, and 5. We further discussed these inflammasomes functions in diseases, including infectious diseases, sepsis, inflammatory autoimmune diseases, and neuroinflammatory diseases.     

DNA hypomethylation leads to cGAS‐induced autoinflammation in the epidermis

DNA methylation is a fundamental epigenetic modification, important across biological processes. The maintenance methyltransferase DNMT1 is essential for lineage differentiation during development, but its functions in tissue homeostasis are incompletely understood. We show that epidermis‐specific DNMT1 deletion severely disrupts epidermal structure and homeostasis, initiating a massive innate immune response and infiltration of immune cells. Mechanistically, DNA hypomethylation in keratinocytes triggered transposon derepression, mitotic defects, and formation of micronuclei. DNA release into the cytosol of DNMT1‐deficient keratinocytes activated signaling through cGAS and STING, thus triggering inflammation. Our findings show that disruption of a key epigenetic mark directly impacts immune and tissue homeostasis, and potentially impacts our understanding of autoinflammatory diseases and cancer immunotherapy.     

Structural and Functional Analyses of DNA-Sensing and Immune Activation by Human cGAS

The detection of cytosolic DNA, derived from pathogens or host cells, by cytosolic receptors is essential for appropriate host immune responses. Cyclic GMP-AMP synthase (cGAS) is a newly identified cytosolic DNA receptor that produces cyclic GMP-AMP, which activates stimulator of interferon genes (STING), resulting in TBK1-IRF3 pathway activation followed by the production of type I interferons. Here we report the crystal structure of human cGAS. The structure revealed that a cluster of lysine and arginine residues forms the positively charged DNA binding surface of human cGAS, which is important for the STING-dependent immune activation. A structural comparison with other previously determined cGASs and our functional analyses suggested that a conserved zinc finger motif and a leucine residue on the DNA binding surface are crucial for the DNA-specific immune response of human cGAS, consistent with previous work. These structural features properly orient the DNA binding to cGAS, which is critical for DNA-induced cGAS activation and STING-dependent immune activation. Furthermore, we showed that the cGAS-induced activation of STING also involves the activation of the NF-κB and IRF3 pathways. Our results indicated that cGAS is a DNA sensor that efficiently activates the host immune system by inducing two distinct pathways.     

α7 Nicotinic Acetylcholine Receptor Signaling Inhibits Inflammasome Activation by Preventing Mitochondrial DNA Release

The mammalian immune system and the nervous system coevolved under the influence of cellular and environmental stress. Cellular stress is associated with changes in immunity and activation of the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome, a key component of innate immunity. Here we show that α7 nicotinic acetylcholine receptor (α7 nAchR)-signaling inhibits inflammasome activation and prevents release of mitochondrial DNA, an NLRP3 ligand. Cholinergic receptor agonists or vagus nerve stimulation significantly inhibits inflammasome activation, whereas genetic deletion of α7 nAchR significantly enhances inflammasome activation. Acetylcholine accumulates in macrophage cytoplasm after adenosine triphosphate (ATP) stimulation in an α7 nAchR-independent manner. Acetylcholine significantly attenuated calcium or hydrogen oxide–induced mitochondrial damage and mitochondrial DNA release. Together, these findings reveal a novel neurotransmitter-mediated signaling pathway: acetylcholine translocates into the cytoplasm of immune cells during inflammation and inhibits NLRP3 inflammasome activation by preventing mitochondrial DNA release.