A flagellin-independent caspase-1 activation pathway that does not require NAIP5 or NRLC4 is induced by the intracellular pathogen Legionella pneumophila . Here we demonstrate that this pathway ...requires caspase-11. Treatment of macrophages with LPS up-regulated the host components required for this caspase-11 activation pathway. Activation by Legionella differed from caspase-11 activation using previously described agonists in that Legionella caspase-11 activation was rapid and required bacteria with a functional type IV secretion system called Dot/Icm. Legionella activation of caspase-11 induced pyroptosis by a mechanism independent of the NAIP/NLRC4 and caspase-1 axis. Legionella activation of caspase-11 stimulated activation of caspase-1 through NLRP3 and ASC. Induction of caspase-11–dependent responses occurred in macrophages deficient in the adapter proteins TRIF or MyD88 but not in macrophages deficient in both signaling factors. Although caspase-11 was produced in macrophages deficient in the type-I IFN receptor, there was a severe defect in caspase-11–dependent pyroptosis in these cells. These data indicate that macrophages respond to microbial signatures to produce proteins that mediate a capsase-11 response and that the caspase-11 system provides an alternative pathway for rapid detection of an intracellular pathogen capable of evading the canonical caspase-1 activation system that responds to bacterial flagellin.
Microarchitectural cues drive aligned fibrillar collagen deposition in vivo and in biomaterial scaffolds, but the cell-signaling events that underlie this process are not well understood. Utilizing a ...multicellular patterning model system that allows for observation of intracellular signaling events during collagen matrix assembly, we investigated the role of calcium (Ca2+) signaling in human mesenchymal stem cells (MSCs) during this process. We observed spontaneous Ca2+ oscillations in MSCs during fibrillar collagen assembly, and hypothesized that the transient receptor potential vanilloid 4 (TRPV4) ion channel, a mechanosensitive Ca2+-permeable channel, may regulate this signaling. Inhibition of TRPV4 nearly abolished Ca2+ signaling at initial stages of collagen matrix assembly, while at later times had reduced but significant effects. Importantly, blocking TRPV4 activity dramatically reduced aligned collagen fibril assembly; conversely, activating TRPV4 accelerated aligned collagen formation. TRPV4-dependent Ca2+ oscillations were found to be independent of pattern shape or subpattern cell location, suggesting this signaling mechanism is necessary for aligned collagen formation but not sufficient in the absence of physical (microarchitectural) cues that force multicellular alignment. As cell-generated mechanical forces are known to be critical to the matrix assembly process, we examined the role of TRPV4-mediated Ca2+ signaling in force generated across the load-bearing focal adhesion protein vinculin within MSCs using an FRET-based tension sensor. Inhibiting TRPV4 decreased tensile force across vinculin, whereas TRPV4 activation caused a dynamic unloading and reloading of vinculin. Together, these findings suggest TRPV4 activity regulates forces at cell-matrix adhesions and is critical to aligned collagen matrix assembly by MSCs.
Caspase-1 activation is a key feature of the innate immune response of macrophages elicited by pathogens and a variety of toxins. Here, we determined the requirement for different adapter proteins ...involved in regulating host processes mediated by caspase-1 after macrophage infection by Legionella pneumophila. The adapter protein Asc was found to be important for caspase-1 activation during L. pneumophila infection. Activation of caspase-1 through Asc did not require the flagellin-sensing pathway involving the host nucleotide-binding domain and leucine-rich repeat-containing protein Ipaf (NLRC4). Asc-dependent caspase-1 activation was inhibited by high extracellular potassium levels, whereas Ipaf-dependent activation was unaffected by potassium treatment. Activation of caspase-1 in macrophages occurred independently of Nalp3 and proteasome activity, suggesting that a previously uncharacterized mechanism for caspase-1 activation through Asc may be triggered by L. pneumophila. Rapid pore formation and pyroptosis induced by L. pneumophila required caspase-1, Ipaf, and bacterial flagellin but occurred independently of Asc. Equivalent levels of active interleukin-18 (IL-18) were detected in the lungs of mice infected with a flagellin-deficient strain of L. pneumophila and Asc-deficient mice infected with wild-type L. pneumophila. Active IL-18 was undetectable in the lungs of Asc-deficient mice infected with an L. pneumophila flagellin mutant, indicating independent roles for Ipaf and Asc in caspase-1-mediated processing and release of IL-18 in vivo. Ipaf-dependent activation of caspase-1 restricted bacterial replication in vivo, whereas Asc was dispensable for restriction of L. pneumophila replication in mice. Thus, L. pneumophila-mediated caspase-1 activation involves the coordinate activities of inflammasomes differentially regulated by Ipaf and Asc.
Control over cellular delivery of different functionalities and their synchronized activation is a challenging task. We report several RNA and RNA/DNA-based nanoparticles designed to conditionally ...activate the RNA interference in various human cells. These nanoparticles allow precise control over their formulation, stability in blood serum, and activation of multiple functionalities. Importantly, interferon and pro-inflammatory cytokine activation assays indicate the significantly lower responses for DNA nanoparticles compared to the RNA counterparts, suggesting greater potential of these molecules for therapeutic use.
Nucleotide-binding domain, leucine-rich repeat containing proteins (NLRs) activate caspase-1 in response to a variety of bacterium-derived signals in macrophages. NLR-mediated activation of caspase-1 ...by Legionella pneumophila occurs through both an NLRC4/NAIP5-dependent pathway and a pathway requiring the adapter protein Asc. Both pathways are needed for maximal activation of caspase-1 and for the release of the cytokines interleukin-1β (IL-1β) and IL-18. Asc is not required for caspase-1-dependent pore formation and cell death induced upon infection of macrophages by L. pneumophila. Here, temporal and spatial localization of caspase-1-dependent processes was examined to better define the roles of Asc and NLRC4 during infection. Imaging studies revealed that caspase-1 localized to a single punctate structure in infected cells containing Asc but not in cells lacking this adapter. Both endogenous Asc and ectopically produced NLRC4 tagged with green fluorescent protein (GFP) were found to localize to caspase-1 puncta following L. pneumophila infection, suggesting that NLRC4 and Asc coordinate signaling through this complex during caspase-1 activation. Formation of caspase-1-containing puncta correlated with caspase-1 processing, suggesting a role for the Asc/NLRC4/caspase-1 complex in caspase-1 cleavage. In cells deficient for Asc, NLRC4 did not assemble into discrete puncta, and pyroptosis occurred at an accelerated rate. These data indicate that Asc mediates integration of NLR components into caspase-1 processing platforms and that recruitment of NLR components into an Asc complex can dampen pyroptotic responses. Thus, a negative feedback role of complexes containing Asc may be important for regulating caspase-1-mediated responses during microbial infection.
Caspase-1 is a protease activated during infection that is central to the regulation of several innate immune pathways. Studies examining the macromolecular complexes containing this protein, known as inflammasomes, have provided insight into the regulation of this protease. This work demonstrates that the intracellular bacterium Legionella pneumophila induces formation of complexes containing caspase-1 by multiple mechanisms and illustrates that an adapter molecule called Asc integrates signals from multiple independent upstream caspase-1 activators in order to assemble a spatially distinct complex in the macrophage. There were caspase-1-associated activities such as cytokine processing and secretion that were controlled by Asc. Importantly, this work uncovered a new role for Asc in dampening a caspase-1-dependent cell death pathway called pyroptosis. These findings suggest that Asc plays a central role in controlling a distinct subset of caspase-1-dependent activities by both assembling complexes that are important for cytokine processing and suppressing processes that mediate pyroptosis.
Control over the simultaneous delivery of different functionalities and their synchronized intracellular activation can greatly benefit the fields of RNA and DNA biomedical nanotechnologies and allow ...for the production of nanoparticles and various switching devices with controllable functions. We present a system of multiple split functionalities embedded in the cognate pairs of RNA-DNA hybrids which are programmed to recognize each other, re-associate and form a DNA duplex while also releasing the split RNA fragments which upon association regain their original functions. Simultaneous activation of three different functionalities (RNAi, Förster resonance energy transfer and RNA aptamer) confirmed by multiple in vitro and cell culture experiments prove the concept. To automate the design process, a novel computational tool that differentiates between the thermodynamic stabilities of RNA-RNA, RNA-DNA and DNA-DNA duplexes was developed. Moreover, here we demonstrate that besides being easily produced by annealing synthetic RNAs and DNAs, the individual hybrids carrying longer RNAs can be produced by RNA polymerase II-dependent transcription of single-stranded DNA templates.
Western (WEEV), eastern (EEEV), and Venezuelan (VEEV) equine encephalitis viruses are mosquito-borne pathogens classified as potential biological warfare agents for which there are currently no ...approved human vaccines or therapies. We aimed to evaluate the safety, tolerability, and immunogenicity of an investigational trivalent virus-like particle (VLP) vaccine, western, eastern, and Venezuelan equine encephalitis (WEVEE) VLP, composed of WEEV, EEEV, and VEEV VLPs.
The WEVEE VLP vaccine was evaluated in a phase 1, randomised, open-label, dose-escalation trial at the Hope Clinic of the Emory Vaccine Center at Emory University, Atlanta, GA, USA. Eligible participants were healthy adults aged 18-50 years with no previous vaccination history with an investigational alphavirus vaccine. Participants were assigned to a dose group of 6 μg, 30 μg, or 60 μg vaccine product and were randomly assigned (1:1) to receive the WEVEE VLP vaccine with or without aluminium hydroxide suspension (alum) adjuvant by intramuscular injection at study day 0 and at week 8. The primary outcomes were the safety and tolerability of the vaccine (assessed in all participants who received at least one administration of study product) and the secondary outcome was immune response measured as neutralising titres by plaque reduction neutralisation test (PRNT) 4 weeks after the second vaccination. This trial is registered at ClinicalTrials.gov, NCT03879603.
Between April 2, 2019, and June 13, 2019, 30 trial participants were enrolled (mean age 32 years, range 21-48; 16 53% female participants and 14 47% male participants). Six groups of five participants each received 6 μg, 30 μg, or 60 μg vaccine doses with or without adjuvant, and all 30 participants completed study follow-up. Vaccinations were safe and well tolerated. The most frequently reported symptoms were mild injection-site pain and tenderness (22 73% of 30) and malaise (15 50% of 30). Dose-dependent differences in the frequency of pain and tenderness were found between the 6 μg, 30 μg, and 60 μg groups (p=0·0217). No significant differences were observed between dosing groups for any other reactogenicity symptom. Two adverse events (mild elevated blood pressure and moderate asymptomatic neutropenia) were assessed as possibly related to the study product in one trial participant (60 μg dose with alum); both resolved without clinical sequelae. 4 weeks after second vaccine administration, neutralising antibodies were induced in all study groups with the highest response seen against all three vaccine antigens in the 30 μg plus alum group (PRNT
geometric mean titre for EEEV 60·8, 95% CI 29·9-124·0; for VEEV 111·5, 49·8-249·8; and for WEEV 187·9, 90·0-392·2). Finally, 4 weeks after second vaccine administration, for all doses, the majority of trial participants developed an immune response to all three vaccine components (24 83% of 29 for EEEV; 26 90% of 29 for VEEV; 27 93% of 29 for WEEV; and 22 76% of 29 for EEEV, VEEV, and WEEV combined).
The favourable safety profile and neutralising antibody responses, along with pressing public health need, support further evaluation of the WEVEE VLP vaccine in advanced-phase clinical trials.
The Vaccine Research Center of the National Institute of Allergy and Infectious Diseases, National Institutes of Health funded the clinical trial. The US Department of Defense contributed funding for manufacturing of the study product.
Dendritic cells (DCs) are specialized phagocytes that internalize exogenous antigens and microbes at peripheral sites, and then migrate to lymphatic organs to display foreign peptides to naïve T ...cells. There are several examples where DCs have been shown to be more efficient at restricting the intracellular replication of pathogens compared to macrophages, a property that could prevent DCs from enhancing pathogen dissemination. To understand DC responses to pathogens, we investigated the mechanisms by which mouse DCs are able to restrict replication of the intracellular pathogen Legionella pneumophila. We show that both DCs and macrophages have the ability to interfere with L. pneumophila replication through a cell death pathway mediated by caspase-1 and Naip5. L. pneumophila that avoided Naip5-dependent responses, however, showed robust replication in macrophages but remained unable to replicate in DCs. Apoptotic cell death mediated by caspase-3 was found to occur much earlier in DCs following infection by L. pneumophila compared to macrophages infected similarly. Eliminating the pro-apoptotic proteins Bax and Bak or overproducing the anti-apoptotic protein Bcl-2 were both found to restore L. pneumophila replication in DCs. Thus, DCs have a microbial response pathway that rapidly activates apoptosis to limit pathogen replication.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The immune system must discriminate between pathogenic and nonpathogenic microbes in order to initiate an appropriate response. Toll-like receptors (TLRs) detect microbial components common to both ...pathogenic and nonpathogenic bacteria, whereas Nod-like receptors (NLRs) sense microbial components introduced into the host cytosol by the specialized secretion systems or pore-forming toxins of bacterial pathogens. The host signaling pathways that respond to bacterial secretion systems remain poorly understood. Infection with the pathogen Legionella pneumophila, which utilizes a type IV secretion system (T4SS), induced an increased proinflammatory cytokine response compared to avirulent bacteria in which the T4SS was inactivated. This enhanced response involved NF-kappaB activation by TLR signaling as well as Nod1 and Nod2 detection of type IV secretion. Furthermore, a TLR- and RIP2-independent pathway leading to p38 and SAPK/JNK MAPK activation was found to play an equally important role in the host response to virulent L. pneumophila. Activation of this MAPK pathway was T4SS-dependent and coordinated with TLR signaling to mount a robust proinflammatory cytokine response to virulent L. pneumophila. These findings define a previously uncharacterized host response to bacterial type IV secretion that activates MAPK signaling and demonstrate that coincident detection of multiple bacterial components enables immune discrimination between virulent and avirulent bacteria.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Transplant candidates can be listed at multiple transplant centers to increase the probability of receiving an organ. We evaluated the association between multilisting (ML) status and access to a ...deceased donor kidney transplant (DDKT) to determine if ML provides a long-term advantage regarding wait-list mortality and recipient outcomes.
Candidates between January 2010 and October 2017 were identified as either singly or multiply listed using Organ Procurement and Transplantation Network data and cohorts before and after implementation of the Kidney Allocation System (KAS). Cross-sectional logistic regression was used to assess relationships between candidate factors and ML prevalence (5.4%).
Factors associated with ML pre-KAS included having blood type B (reference, type O; odds ratio OR, 1.20; P < .001), having private insurance (OR, 1.5; P < .001), wait time (OR, 1.28; P < .001), and increasing calculated panel-reactive antibody (cPRA) (reference, cPRA 0-100; OR for cPRA 80-98, 2.83; OR for cPRA 99, 3.47; OR for cPRA 100, 5.18; P < .001). Transplant rates were double for multilisted vs singly listed recipients (adjusted hazard ratio aHR, 2.16; P < .001). Extra–donor service area ML candidates received transplants 2.5 years quicker than single-listing (SL) candidates, conferring a 42% wait-list advantage. Recipient death (aHR, 0.94; P = .122) and graft failure (aHR, 0.91; P = .006) rates were also lower for ML recipients.
In the KAS era, ML continues to increase the likelihood of receiving a DDKT and lower the incidence of wait-list mortality, and it confers a survival advantages over SL.
•Transplant candidates can multiply list to increase the probability of receiving an organ. We evaluated if this provides a long-term advantage and improves recipient outcomes.•Multiply listed candidates before and after implementation of the Kidney Allocation System (KAS) were analyzed.•Transplant rates in multilisted recipients were double those of singly listed recipients.•Patient death and graft failure rates were also lower for multilisting recipients.•In the KAS era, multiple listing confers survival advantages over single listing.
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