Inflammasomes are central mediators of host defense to a wide range of microbial pathogens. The NLRP3 inflammasome plays a key role in triggering caspase-1 dependent IL-1β maturation and resistance to fungal dissemination in Candida albicans infection. β-glucans are major components of fungal cell walls that trigger IL-1β secretion in both murine and human immune cells. In this study, we sought to determine the contribution of β-glucans to C. albicans-induced inflammasome responses in mouse dendritic cells. We show that the NLRP3-ASC-caspase-1 inflammasome is absolutely critical for IL-1β production in response to β-glucans. Interestingly, we also found that both Complement Receptor 3 (CR3/Mac-1) and dectin-1 play a crucial role in coordinating β-glucan-induced IL-1β processing as well as a cell death response. In addition to the essential role of caspase-1, we identify an important role for the pro-apoptotic protease caspase-8 in promoting β-glucan-induced cell death and NLRP3 inflammasome-dependent IL-1β maturation. A strong requirement for Complement Receptor 3 and caspase-8 was also found for NLRP3 dependent IL-1β production in response to heat killed Candida albicans. Together, these results define the importance of dectin-1, CR3 and caspase-8, in addition to the canonical NLRP3 inflammasome, in mediating β-glucan and C. albicans induced innate responses in dendritic cells. Collectively, these findings establish a novel link between β-glucan recognition receptors and the inflammatory proteases caspase-8 and caspase-1 in coordinating cytokine secretion and cell death in response to immunostimulatory fungal components.
TLR2 promotes NLRP3 inflammasome activation via an early MyD88-IRAK1-dependent pathway that provides a priming signal (signal 1) necessary for activation of the inflammasome by a second potassium-depleting signal (signal 2). Here we show that TLR3 binding to dsRNA promotes post-translational inflammasome activation through intermediate and late TRIF/RIPK1/FADD-dependent pathways. Both pathways require the scaffolding but not the catalytic function of caspase-8 or RIPK1. Only the late pathway requires kinase competent RIPK3 and MLKL function. Mechanistically, FADD/caspase-8 scaffolding function provides a post-translational signal 1 in the intermediate pathway, whereas in the late pathway it helps the oligomerization of RIPK3, which together with MLKL provides both signal 1 and 2 for inflammasome assembly. Cytoplasmic dsRNA activates NLRP3 independent of TRIF, RIPK1, RIPK3 or mitochondrial DRP1, but requires FADD/caspase-8 in wildtype macrophages to remove RIPK3 inhibition. Our study provides a comprehensive analysis of pathways that lead to NLRP3 inflammasome activation in response to dsRNA.
Inflammasome activation requires a complex and incompletely understood network of signalling events. Here the authors characterize step-by-step contributions of TLR3, caspase-8, RIPK3 and MLKL to the activation of NLRP3 inflammasome in response to double-stranded RNA.
The four Toll/IL-1R (TIR) domain-containing adaptor proteins MyD88, MAL, TRIF and TRAM are well established as essential mediators of TLR signaling and gene induction following microbial detection. In contrast, the function of the fifth, most evolutionarily conserved TIR adaptor sterile alpha and HEAT/Armadillo motif-containing protein (SARM) has remained more elusive. Recent studies of Sarm−/− mice have highlighted a role for SARM in stress-induced neuronal cell death and immune responses in the CNS. However, whether SARM has a role in immune responses in peripheral myeloid immune cells is less clear. Thus, we characterized TLR-induced cytokine responses in SARM-deficient murine macrophages, and discovered a requirement for SARM in CCL5 production, while gene induction of TNF, IL1β, CCL2 and CXCL10 were SARM-independent. SARM was not required for TLR-induced activation of MAPKs or of transcription factors implicated in CCL5 induction, namely NF-κB and IFN regulatory factors, nor for Ccl5 mRNA stability or splicing. However, SARM was critical for the recruitment of transcription factors and of RNA polymerase II to the Ccl5 promoter. Strikingly, the requirement of SARM for CCL5 induction was not restricted to TLR pathways, as it was also apparent in cytosolic RNA and DNA responses. Thus, this study identifies a new role for SARM in CCL5 expression in macrophages.
The inflammasomes are multiprotein complexes that activate caspase-1 in response to infections and stress, resulting in the secretion of pro-inflammatory cytokines. Here we report that IKKα is a critical negative regulator of ASC-dependent inflammasomes. IKKα controls the inflammasome at the level of the adaptor ASC, which interacts with IKKα in the nucleus of resting macrophages in an IKKα kinase-dependent manner. Loss of IKKα kinase activity results in inflammasome hyperactivation. Mechanistically, the downstream nuclear effector IKKi facilitates translocation of ASC from the nucleus to the perinuclear area during inflammasome activation. ASC remains under the control of IKKα in the perinuclear area following translocation of the ASC/IKKα complex. Signal 2 of NLRP3 activation leads to inhibition of IKKα kinase activity through the recruitment of PP2A, allowing ASC to participate in NLRP3 inflammasome assembly. Taken together, these findings reveal a IKKi-IKKα-ASC axis that serves as a common regulatory mechanism for ASC-dependent inflammasomes.
An inducible program of inflammatory gene expression is central to anti-microbial defenses. Signal-dependent activation of transcription factors, transcriptional co-regulators and chromatin modifying factors collaborate to control this response. Here we identify a long noncoding RNA that acts as a key regulator of this inflammatory response. Germline-encoded receptors such as the Toll-like receptors induce the expression of numerous lncRNAs. One of these, lincRNA-Cox2 mediates both the activation and repression of distinct classes of immune genes. Transcriptional repression of target genes is dependent on interactions of lincRNA-Cox2 with heterogeneous nuclear ribonucleoprotein A/B and A2/B1. Collectively, these studies unveil a central role of lincRNA-Cox2 as a broad acting regulatory component of the circuit that controls the inflammatory response.
Stimulator of interferon genes (STING, also named MITA, MYPS or ERIS) is an intracellular DNA sensor that induces type I interferon through its interaction with TANK-binding kinase 1 (TBK1). Here we found that the nucleotide-binding, leucine-rich repeat containing protein, NLRC3, reduced STING-dependent innate immune activation in response to cytosolic DNA, cyclic di-GMP (c-di-GMP) and DNA viruses. NLRC3 associated with both STING and TBK1, and impeded STING-TBK1 interaction and downstream type I interferon production. Using purified recombinant proteins NLRC3 was found to interact directly with STING. Furthermore, NLRC3 prevented proper trafficking of STING to perinuclear and punctated region, known to be important for its activation. In animals, herpes simplex virus 1 (HSV-1)-infected Nlrc3−/− mice exhibited enhanced innate immunity, reduced morbidity and viral load. This demonstrates the intersection of two key pathways of innate immune regulation, NLR and STING, to fine tune host response to intracellular DNA, DNA virus and c-di-GMP
The TLR4 ligand LPS causes mouse B cells to undergo IgE and IgG1 isotype switching in the presence of IL-4. TLR4 activates two signaling pathways mediated by the adaptor molecules MyD88 and TRAM, which recruits TRIF. Following stimulation with LPS+IL-4, Tram−/− and Trif−/− B cells completely failed to express Cε germ line transcripts (GLT) and secrete IgE. In contrast, Myd88−/− B cells had normal expression of Cε GLT, but reduced IgE secretion in response to LPS+IL-4. Following LPS+IL-4 stimulation, Cγ1 GLT expression was modestly reduced in Tram−/− and Trif−/− B cells, whereas Aicda expression and IgG1 secretion were reduced in Tram−/−, Trif−/−, and Myd88−/− B cells. B cells from all strains secreted normal amounts of IgE and IgG1 in response to anti-CD40+IL-4. Following stimulation with LPS+IL-4, Trif−/− B cells failed to sustain NFκB p65 nuclear translocation beyond 3 hours and had reduced binding of p65 to the Iε promoter. Addition of the NFκB inhibitor, JSH-23, to wild-type B cells 15 hours after LPS+IL-4 stimulation selectively blocked Cε GLT expression and IgE secretion, but had little effect on Cγ1 GLT expression and IgG secretion. These results indicate that sustained activation of NFκB driven by TRIF is essential for LPS+IL-4 driven activation of the Cε locus and class switching to IgE.
B cells; IgE; LPS; TLR4; TRAM; TRIF
Inflammasomes elicit host defense inside cells by activating caspase-1 for cytokine maturation and cell death. AIM2 and NLRP3 are representative sensor proteins in two major families of inflammasomes. The adaptor protein ASC bridges the sensor proteins and caspase-1 to form ternary inflammasome complexes, achieved through pyrindomain (PYD) interactions between sensors and ASC, and caspase activation and recruitment domain (CARD) interactions between ASC and caspase-1. We found that PYD and CARD both form filaments. Activated AIM2 and NLRP3 nucleate PYD filaments of ASC, which in turn cluster the CARD of ASC. ASC thus nucleates CARD filaments of caspase-1 leading to proximity-induced activation. Endogenous NLRP3 inflammasome is also filamentous. The cryo-EM structure of ASCPYD filament at near-atomic resolution provides a template for homo- and hetero-PYD/PYD associations, as confirmed by structure-guided mutagenesis. We propose that ASC-dependent inflammasomes in both families share a unified assembly mechanism that involves two successive steps of nucleation-induced polymerization.
The transcriptional repressor BLIMP1 is a master regulator of B and T cell differentiation. To examine the role of BLIMP1 in innate immunity we used a conditional knockout (CKO) of Blimp1 in myeloid cells and found that Blimp1 CKO mice were protected from lethal infection induced by Listeria monocytogenes. Transcriptome analysis of Blimp1 CKO macrophages identified the murine chemokine (C-C motif) ligand 8, CCL8 as a direct target of Blimp1-mediated transcriptional repression in these cells. BLIMP1-deficient macrophages expressed elevated levels of Ccl8 and consequently Blimp1 CKO mice had higher levels of circulating CCL8 resulting in increased neutrophils in the peripheral blood, promoting a more aggressive anti-bacterial response. Mice lacking the Ccl8 gene were more susceptible to L. monocytogenes infection than wild type mice. While CCL8 failed to recruit neutrophils directly, it was chemotactic for γ/δ T cells and CCL8-responsive γ/δ T cells were enriched for IL-17F. Finally, CCL8-mediated enhanced clearance of L. monocytogenes was dependent on γ/δ T cells. Collectively, these data reveal an important role for BLIMP1 in modulating host-defenses by suppressing expression of the chemokine CCL8.
Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are essential intracellular detectors of viral RNA. They contribute to the type I interferon (IFN) response that is crucial for host defense against viral infections. Given the potent antiviral and proinflammatory activities elicited by the type I IFNs, induction of the type I IFN response is tightly regulated. Members of the tripartite motif (TRIM) family of proteins have recently emerged as key regulators of antiviral immunity. We show that TRIM13, an E3 ubiquitin ligase, is expressed in immune cells and is upregulated in bone marrow-derived macrophages upon stimulation with inducers of type I IFN. TRIM13 interacts with MDA5 and negatively regulates MDA5-mediated type I IFN production in vitro, acting upstream of IFN regulatory factor 3. We generated Trim13−/− mice and show that upon lethal challenge with encephalomyocarditis virus (EMCV), which is sensed by MDA5, Trim13−/− mice produce increased amounts of type I IFNs and survive longer than wild-type mice. Trim13−/− murine embryonic fibroblasts (MEFs) challenged with EMCV or poly(I·C) also show a significant increase in beta IFN (IFN-β) levels, but, in contrast, IFN-β responses to the RIG-I-detected Sendai virus were diminished, suggesting that TRIM13 may play a role in positively regulating RIG-I function. Together, these results demonstrate that TRIM13 regulates the type I IFN response through inhibition of MDA5 activity and that it functions nonredundantly to modulate MDA5 during EMCV infection.
IMPORTANCE The type I interferon (IFN) response is crucial for host defense against viral infections, and proper regulation of this pathway contributes to maintaining immune homeostasis. Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are intracellular detectors of viral RNA that induce the type I IFN response. In this study, we show that expression of the gene tripartite motif 13 (Trim13) is upregulated in response to inducers of type I IFN and that TRIM13 interacts with both MDA5 and RIG-I in vitro. Through the use of multiple in vitro and in vivo model systems, we show that TRIM13 is a negative regulator of MDA5-mediated type I IFN production and may also impact RIG-I-mediated type I IFN production by enhancing RIG-I activity. This places TRIM13 at a key junction within the viral response pathway and identifies it as one of the few known modulators of MDA5 activity.
Inflammasome activation is gaining recognition as an important mechanism for protection during viral infection. Here, we investigate whether Rift Valley fever virus, a negative-strand RNA virus, can induce inflammasome responses and IL-1β processing in immune cells. We have determined that RVFV induces NLRP3 inflammasome activation in murine dendritic cells, and that this process is dependent upon ASC and caspase-1. Furthermore, absence of the cellular RNA helicase adaptor protein MAVS/IPS-1 significantly reduces extracellular IL-1β during infection. Finally, direct imaging using confocal microscopy shows that the MAVS protein co-localizes with NLRP3 in the cytoplasm of RVFV infected cells.
inflammasome; NLRP3; ASC; caspase-1; Rift Valley fever virus; virus; IL-1β; dendritic cells; murine
Hemozoin (Hz) is the crystalline detoxification product of hemoglobin in plasmodial-infected erythrocytes. We previously proposed that Hz can carry plasmodial DNA into a subcellular compartment accessible to Toll-like receptor 9 (TLR9), inducing an inflammatory signal. Hemozoin also activates the NLRP3 inflammasome in primed cells. We found that Hz appears to co localize with DNA in infected erythrocytes, even prior to RBC rupture or phagolysosomal digestion. Using synthetic Hz coated in vitro with plasmodial genomic DNA (gDNA) or CpG-oligonucleotides, we observed that DNA-complexed Hz induced TLR9 translocation, providing a priming and an activation signal for inflammasomes. After phagocytosis, Hz and DNA dissociate. Hz subsequently induces phagolysosomal destabilization, allowing phagolysosomal contents access to the cytosol where DNA receptors become activated. Similar observations were made with plasmodial-infected RBC. Finally, infected erythrocytes activated both the NLRP3 and AIM2 inflammasomes. These observations suggest that Hz and DNA work together to induce systemic inflammation during malaria.
Autophagy has been implicated as a component of host defense, but the significance of antimicrobial autophagy in vivo and the mechanism by which it is regulated during infection are poorly defined. Here we found that antiviral autophagy was conserved in flies and mammals during infection with Rift Valley fever virus (RVFV), a mosquito-borne virus that causes disease in humans and livestock. In Drosophila, Toll-7 limited RVFV replication and mortality through activation of autophagy. RVFV infection also elicited autophagy in mouse and human cells, and viral replication was increased in the absence of autophagy genes. The mammalian Toll-like receptor adaptor, MyD88, was required for anti-RVFV autophagy, revealing an evolutionarily conserved requirement for pattern-recognition receptors in antiviral autophagy. Pharmacologic activation of autophagy inhibited RVFV infection in mammalian cells, including primary hepatocytes and neurons. Thus, autophagy modulation might be an effective strategy for treating RVFV infection, which lacks approved vaccines and therapeutics.
Loss of function mutations in the Fas death receptor or its ligand result in a lymphoproliferative syndrome and exacerbate clinical disease in most lupus-prone strains of mice. One exception is mice injected with 2,6,10,14-Tetramethylpentadecane (TMPD), a hydrocarbon oil commonly known as pristane, which induces SLE-like disease. While Fas/FasL interactions have been strongly implicated in the activation induced cell death of both lymphocytes and other antigen presenting cells, FasL can also trigger the production of pro-inflammatory cytokines. FasL is a transmembrane protein with a matrix metalloproteinase (MMP) cleavage site in the ectodomain. MMP cleavage inactivates membrane-bound FasL (mFasL) and releases a soluble form, sFasL, reported to have both antagonist and agonist activity. To better understand the impact of FasL cleavage on both the pro-apoptotic and proinflammatory activity of FasL, its cleavage site was deleted through targeted mutation, to produce the ΔCS mouse line. ΔCS mice express higher levels of mFasL than WT mice and fail to release sFasL. To determine to what extent FasL promotes inflammation in lupus mice, TMPD-injected FasL-deficient and ΔCS BALB/c mice were compared to control TMPD-injected BALB/c mice. We found that FasL-deficiency significantly reduced the early inflammatory exudate induced by TMPD injection. By contrast, ΔCS mice developed a markedly exacerbated disease profile associated with a higher frequency of splenic neutrophils and macrophages, a profound change in ANA specificity, and markedly increased proteinuria and kidney pathology, compared to controls. These results demonstrate that FasL promotes inflammation in TMPD-induced autoimmunity, and its cleavage limits FasL pro-inflammatory activity.
Activation of the NLRP3 inflammasome by diverse stimuli requires a priming signal from Toll-like receptors (TLRs) and an activation signal from purinergic receptors or pore-forming toxins. Here we demonstrate through detailed analysis of NLRP3 activation in macrophages deficient in key downstream TLR signaling molecules that MyD88 is required for an immediate early phase, whereas TRIF is required for a subsequent intermediate phase of posttranslational NLRP3 activation. Both IRAK1 and IRAK4 kinases are critical for rapid activation of NLRP3 through the MyD88 pathway, but only IRAK1 is partially required in the TRIF pathway. IRAK1 and IRAK4 are also required for rapid activation of NLRP3 by Listeria monocytogenes as deletion of IRAK1 or IRAK4 led to defective inflammasome activation. These findings define the pathways that lead to rapid NLRP3 activation and identify IRAK1 as a critical mediator of a transcription-independent, inflammasome-dependent early warning response to pathogenic infection.
Mycobacterium tuberculosis (Mtb) extracellular DNA (eDNA) gains access to the host cell cytosol via the ESX-1 secretion system. It is puzzling that this eDNA of Mtb does not induce activation of the AIM2-inflammasome since AIM2 recognizes cytosolic DNA. Here we show that non-virulent mycobacteria such as M. smegmatis induce AIM2-inflammasome activation, which is dependent upon their strong induction of IFN-β production. In contrast, Mtb, but not an ESX-1 deficient mutant, inhibits the AIM2-inflammasome activation induced by either M. smegmatis or transfected dsDNA. The inhibition does not involve changes in host cell AIM2 mRNA or protein levels but led to decreased activation of caspase-1. We furthermore demonstrate that Mtb inhibits IFN-β production and signaling, which was partially responsible for the inhibition of AIM2 activation. In conclusion, we report a novel immune evasion mechanism of Mtb that involves the ESX-1-dependent, direct or indirect, suppression of the host cell AIM2-inflammasome activation during infection.
Synthetic oligodeoxynucleotides comprised of the immunosuppressive motif TTAGGG block TLR9 signaling, prevent STAT1 and STAT4 phosphorylation and attenuate a variety of inflammatory responses in vivo. Here, we demonstrate that such suppressive oligodeoxynucleotides (sup ODN) abrogate activation of cytosolic nucleic acid sensing pathways. Pretreatment of dendritic cells and macrophages with the suppressive ODN-A151 abrogated type I IFN, TNFα and ISG induction in response to cytosolic dsDNA. In addition, A151 abrogated caspase-1-dependent IL-1β and IL-18 maturation in dendritic cells stimulated with dsDNA and murine cytomegalovirus (MCMV). Inhibition was dependent on A151’s phosphorothioate backbone while substitution of the guanosine residues for adenosine negatively affected potency. A151 mediates these effects by binding to AIM2 in a manner that is competitive with immune-stimulatory DNA and as a consequence prevents AIM2 inflammasome complex formation. Collectively, these findings reveal a new route by which suppressive ODNs modulate the immune system and unveil novel applications for suppressive ODNs in the treatment of infectious and autoimmune diseases.
Measurement of protease activity in living cells or organisms remains a challenging task. We here present a transgene-encoded biosensor that reports the proteolytic activity of caspase-1 in the course of inflammasome activation and that of other proteases in a highly sensitive and specific manner. This protease reporter is based on the biological activity of a pro-interleukin (IL)-1β-Gaussia luciferase (iGLuc) fusion construct, in which pro-IL-1β-dependent formation of protein aggregates renders GLuc enzyme inactive. Cleavage leads to monomerization of this biosensor protein, resulting in a strong gain in luciferase activity. Exchange of the canonical caspase-1 cleavage site in this reporter construct allows the generation of protease biosensors with additional specificities. The high sensitivity, signal-to-background ratio and specificity of the iGLuc system renders it a useful tool to study proteolytic events in mouse and human cells at high throughput and to monitor protease activity in mice in vivo.
Autophagosomes delivers cytoplasmic constituents to lysosomes for degradation while inflammasomes are molecular platforms activated by infection or stress that regulate the activity of caspase-1 and the maturation of interleukin 1β (IL-1β) and IL-18. Here we show that the induction of AIM2 or NLRP3 inflammasomes in macrophages triggered RalB activation and autophagosome formation. The induction of autophagy did not depend upon ASC or capase-1, but was dependent on the presence of the inflammasome sensor. Blocking autophagy potentiated inflammasome activity while stimulating autophagy limited it. Assembled inflammasomes underwent ubiquitination and recruited the autophagic adaptor p62, which assisted their delivery to autophagosomes. Our data indicate that autophagy accompanies inflammasome activation to temper inflammation by eliminating active inflammasomes.
inflammasome; macrophage; autophagy; signal transduction; ubiquitination
The nucleotidyl transferase cGAS, its second messenger product cGAMP and the cGAMP sensor STING, form the basic mechanism of DNA sensing in the cytoplasm of mammalian cells. Several new reports now uncover key structural features associated with DNA recognition by cGAS and the catalytic mechanisms of cGAMP generation. Concurrent studies also reveal unique phosphodiester linkages in endogenous cGAMP that distinguish it from microbial cGAMP and other cyclic-di-nucleotides. Together, these studies provide a new perspective on DNA recognition in the innate immune system.
Before they infect red blood cells and cause malaria, Plasmodium parasites undergo an obligate and clinically silent expansion phase in the liver that is supposedly undetected by the host. Here, we demonstrate the engagement of a type I interferon (IFN) response during Plasmodium replication in the liver. We identified Plasmodium RNA as a novel pathogen-associated molecular pattern (PAMP) capable of activating a type I IFN response via the cytosolic pattern recognition receptor Mda5. This response, initiated by liver-resident cells through the adaptor molecule for cytosolic RNA sensors, Mavs, and the transcription factors Irf3 and Irf7, is propagated by hepatocytes in an interferon-α/β receptor–dependent manner. This signaling pathway is critical for immune cell–mediated host resistance to liver-stage Plasmodium infection, which can be primed with other PAMPs, including hepatitis C virus RNA. Together, our results show that the liver has sensor mechanisms for Plasmodium that mediate a functional antiparasite response driven by type I IFN.
Obesity is associated with the development of asthma and considerable asthma-related healthcare utilization. To understand the immunological pathways that lead to obesity-associated asthma, we fed mice a high fat diet for 12 weeks, which resulted in obesity and the development of airway hyperreactivity (AHR), a cardinal feature of asthma. This AHR depended on innate immunity, since it occurred in obese Rag−/− mice, and on IL-17A and the NLRP3 inflammasome, since it did not develop in obese Il17−/− or Nlrp3−/− mice. The AHR was also associated with the presence in the lungs of CCR6+ innate lymphoid cells producing IL-17A (ILC3 cells), which could by themselves mediate AHR when adoptively transferred into Rag2−/−
Il2rγ−/− mice. IL-1β played an important role by expanding the ILC3 cells, and treatment to block the function of IL-1β abolished obesity-induced AHR. Since we found ILC3-like cells in the bronchoalveolar lavage fluid of human patients with asthma, we suggest that obesity-associated asthma is facilitated by inflammation mediated by NLRP3, IL-1β and ILC3 cells.
airway hyperreactivity; asthma; obesity; innate lymphoid cells; IL-17; NLRP3; ILC3
Vascular disrupting agents (VDAs) such as DMXAA (5,6-dimethylxanthenone-4-acetic acid) represent a novel approach for cancer treatment. DMXAA has potent anti-tumor activity in mice and, despite significant pre-clinical promise, failed human clinical trials. The anti-tumor activity of DMXAA has been linked to its ability to induce type I interferons in macrophages although the molecular mechanisms involved are poorly understood. Here we identify STING as a direct receptor for DMXAA leading to TBK1 and IRF3 signaling. Remarkably, the ability to sense DMXAA was restricted to murine STING. Human STING failed to bind to or signal in response to DMXAA. Human STING also failed to signal in response to cyclic-dinucleotides, conserved bacterial second messengers known to bind and activate murine STING signaling. Collectively, these findings detail an unexpected species-specific role for STING as a receptor for an anti-cancer drug and uncover important insights that may explain the failure of DMXAA in clinical trials for human cancer.
Nucleotide-binding oligomerization domain-like receptors (NLRs) detect pathogens and danger-associated signals within the cell. Salmonella Typhimurium, an intracellular pathogen, activates caspase-1 required for the processing of the pro-inflammatory cytokines, pro-IL-1β and pro-IL-18, and pyroptosis. Here we show that Salmonella infection induces the formation of an ASC–Caspase-8–Caspase-1 inflammasome in macrophages. Caspase-8 and caspase-1 are recruited to the ASC focus independently of one other. Salmonella infection initiates caspase-8 proteolysis in a manner dependent on NLRC4 and ASC, but not NLRP3, caspase-1 or caspase-11. Caspase-8 primarily mediates the synthesis of pro-IL-1β, but is dispensable for Salmonella-induced cell death. Overall, our findings highlight that the ASC inflammasome can recruit different members of the caspase family to induce distinct effector functions in response to Salmonella infection.
The innate immune system is important for control of infections, including herpesvirus infections. Intracellular DNA potently stimulates antiviral IFN responses. It is known that plasmacytoid dendritic cells sense herpesvirus DNA in endosomes via TLR9, and that non-immune tissue cells can sense herpesvirus DNA in the nucleus. However, it remains unknown how and where myeloid cells, like macrophages and conventional dendritic cells, detect infections with herpesviruses. Here we demonstrate that the HSV-1 capsid was ubiquitinated in the cytosol and degraded by the proteasome, hence releasing genomic DNA into the cytoplasm for detection by DNA sensors. In this context, the DNA sensor IFI16 is important for induction of IFN-β in human macrophages after infection with HSV-1 and CMV. Viral DNA localized to the same cytoplasmic regions as IFI16, with DNA sensing being independent of viral nuclear entry. Thus, proteasomal degradation of herpesvirus capsids releases DNA to the cytoplasm for recognition by DNA sensors.