Invasive pulmonary aspergillosis causes substantial mortality in immunocompromised individuals. Recognition of Aspergillus fumigatus by the host immune system leads to activation of the inflammasome, ...which provides protection against infection. However, regulation of inflammasome activation at the molecular level is poorly understood. Here, we describe two distinct pathways that coordinately control inflammasome activation during A. fumigatus infection. The C-type lectin receptor pathway activates both MAPK and NF-κB signalling, which leads to induction of downstream mediators, such as the transcription factor IRF1, and also primes the inflammasomes. Toll-like receptor signalling through the adaptor molecules MyD88 and TRIF in turn mediates efficient activation of IRF1, which induces IRGB10 expression. IRGB10 targets the fungal cell wall, and the antifungal activity of IRGB10 causes hyphae damage, modifies the A. fumigatus surface and inhibits fungal growth. We also demonstrate that one of the major fungal pathogen-associated molecular patterns, β-glucan, directly triggers inflammasome assembly. Thus, the concerted activation of both Toll-like receptors and C-type lectin receptors is required for IRF1-mediated IRGB10 regulation, which is a key event governing ligand release and inflammasome activation upon A. fumigatus infection.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The integrated stress response (ISR) regulates cellular homeostasis and cell survival following exposure to stressors. Cell death processes such as apoptosis and pyroptosis are known to be modulated ...by stress responses, but the role of the ISR in necroptosis is poorly understood. Necroptosis is an inflammatory, lytic form of cell death driven by the RIPK3-MLKL signaling axis. Here, we show that macrophages that have induced the ISR are protected from subsequent necroptosis. Consistent with a reduction in necroptosis, phosphorylation of RIPK1, RIPK3, and MLKL is reduced in macrophages pre-treated with ISR-inducing agents that are challenged with necroptosis-inducing triggers. The stress granule component DDX3X, which is involved in ISR-mediated regulation of pyroptosis, is not required for protecting ISR-treated cells from necroptosis. Disruption of stress granule assembly or knockdown of
restored necroptosis in pre-stressed cells. Together, these findings identify a critical role for the ISR in limiting necroptosis in macrophages.
Bacteria require at least one pathway to rescue ribosomes stalled at the ends of mRNAs. The primary pathway for ribosome rescue is trans-translation, which is conserved in >99% of sequenced bacterial ...genomes. Some species also have backup systems, such as ArfA or ArfB, which can rescue ribosomes in the absence of sufficient trans-translation activity. Small-molecule inhibitors of ribosome rescue have broad-spectrum antimicrobial activity against bacteria grown in liquid culture. These compounds were tested against the tier 1 select agent Francisella tularensis to determine if they can limit bacterial proliferation during infection of eukaryotic cells. The inhibitors KKL-10 and KKL-40 exhibited exceptional antimicrobial activity against both attenuated and fully virulent strains of F. tularensis in vitro and during ex vivo infection. Addition of KKL-10 or KKL-40 to macrophages or liver cells at any time after infection by F. tularensis prevented further bacterial proliferation. When macrophages were stimulated with the proinflammatory cytokine gamma interferon before being infected by F. tularensis, addition of KKL-10 or KKL-40 reduced intracellular bacteria by >99%, indicating that the combination of cytokine-induced stress and a nonfunctional ribosome rescue pathway is fatal to F. tularensis Neither KKL-10 nor KKL-40 was cytotoxic to eukaryotic cells in culture. These results demonstrate that ribosome rescue is required for F. tularensis growth at all stages of its infection cycle and suggest that KKL-10 and KKL-40 are good lead compounds for antibiotic development.
Interleukin-1 receptor-deficient (IL-1R⁻/⁻) mice are healthy despite being colonized by commensal microbes but are defective in defenses against specific pathogens, suggesting that IL-1R-mediated ...effects contribute to immune responses against specific pathogenic mechanisms. To better define the role of IL-1R in immunity to respiratory infections, we challenged IL-1R⁻/⁻ mice with Bordetella pertussis and Bordetella parapertussis, the causative agents of whooping cough. Following inoculation with B. pertussis, but not B. parapertussis, IL-1R⁻/⁻ mice showed elevated bacterial numbers and more extensive inflammatory pathology than wild-type mice. Acellular B. pertussis vaccines were not efficiently protective against B. pertussis in IL-1R⁻/⁻ mice. B. pertussis-stimulated dendritic cells from IL-1R⁻/⁻ mice produced higher levels of tumor necrosis factor alpha (TNF-α) and IL-6 than wild-type cells. Moreover, elevated levels of gamma interferon (IFN-γ) and TNF-α but lower levels of IL-10 were detected during B. pertussis infection in IL-1R⁻/⁻ mice. Since B. parapertussis did not cause severe disease in IL-1R⁻/⁻ mice, we hypothesized that the extreme requirement for IL-1R involves pertussis toxin (Ptx), which is expressed only by B. pertussis. An isogenic Ptx-deficient B. pertussis strain had only a modest phenotype in wild-type mice but was completely defective in causing lethal disease in IL-1R⁻/⁻ mice, indicating that the particular virulence of B. pertussis in these mice requires Ptx. Ptx contributes to IL-1β induction by B. pertussis, which is involved in IL-10 induction through IL-1R signaling. IL-10 treatment reduced B. pertussis numbers in IL-1R⁻/⁻ mice, suggesting that the lower IL-10 responses partially account for the uncontrolled inflammation and bacterial growth in these mice.
Background
Retrospective analysis of patients undergoing cancer surgery suggests the use of regional anesthesia may reduce cancer recurrence and improve survival. Amide-linked local anesthetics have ...antiinflammatory properties, although the mechanism of action in this regard is unclear. As inflammatory processes involving Src tyrosine protein kinase and intercellular adhesion molecule-1 are important in tumor growth and metastasis, we hypothesized that amide-linked local anesthetics may inhibit inflammatory Src-signaling involved in migration of adenocarcinoma cells.
Methods
NCI-H838 lung cancer cells were incubated with tumor necrosis factor-α in absence/presence of ropivacaine, lidocaine, or chloroprocaine (1 nM-100 μM). Cell migration and total cell lysate Src-activation and intercellular adhesion molecule-1 phosphorylation were assessed. The role of voltage-gated sodium-channels in the mechanism of local anesthetic effects was also evaluated.
Results
Ropivacaine treatment (100 μM) of H838 cells for 20 min decreased basal Src activity by 62% (P=0.003), and both ropivacaine and lidocaine coadministered with tumor necrosis factor-α statistically significantly decreased Src-activation and intercellular adhesion molecule-1 phosphorylation, whereas chloroprocaine had no such effect. Migration of these cells at 4 h was inhibited by 26% (P=0.005) in presence of 1 μM ropivacaine and 21% by 1 μM lidocaine (P=0.004). These effects of ropivacaine and lidocaine were independent of voltage-gated sodium-channel inhibition.
Conclusions
This study indicates that amide-, but not ester-linked, local anesthetics may provide beneficial antimetastatic effects. The observed inhibition of NCI-H838 cell migration by lidocaine and ropivacaine was associated with the inhibition of tumor necrosis factor-α-induced Src-activation and intercellular adhesion molecule-1 phosphorylation, providing the first evidence of a molecular mechanism that appears to be independent of their known role as sodium-channel blockers.
Progress towards a safe and effective vaccine for the prevention of tularemia has been hindered by a lack of knowledge regarding the correlates of protective adaptive immunity and a lack of tools to ...generate this knowledge. CD8.sup.+ T cells are essential for protective immunity against virulent strains of Francisella tularensis, but to-date, it has not been possible to study these cells in an antigen-specific manner. Here, we report the development of a tool for expression of the model antigen ovalbumin (OVA) in F. tularensis, which allows for the study of CD8.sup.+ T cell responses to the bacterium. We demonstrate that in response to intranasal infection with the F. tularensis Live Vaccine Strain, adoptively transferred OVA-specific CD8.sup.+ T cells expand after the first week and produce IFN-gamma but not IL-17. Effector and central memory subsets develop with disparate kinetics in the lungs, draining lymph node and spleen. Notably, OVA-specific cells are poorly retained in the lungs after clearance of infection. We also show that intranasal vaccination leads to more antigen-specific CD8.sup.+ T cells in the lung-draining lymph node compared to scarification vaccination, but that an intranasal booster overcomes this difference. Together, our data show that this novel tool can be used to study multiple aspects of the CD8.sup.+ T cell response to F. tularensis. Use of this tool will enhance our understanding of immunity to this deadly pathogen.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Uveal melanoma (UM) is the most common form of ocular cancer in adults and metastatic disease currently has no effective therapy. Over 80% of UM tumors harbor activating mutations in the ...heterotrimeric G protein α subunits GNAQ or GNA11. These mutations induce protein kinase C (PKC) and the MAP kinase pathway, which signals through RAF, MEK, and ERK. Clinical trials of MEK and PKC inhibitors are underway to treat UM, however, novel therapeutic strategies may be required for effective treatment. To identify rational combination therapies in the context of MEK or PKC inhibition, we performed a pooled, genome-wide synthetic lethal RNA interference screen. Candidate genes whose silencing sensitizes GNAQ/11-mutant UM cells to MEK or PKC inhibition were nominated and further validated across a panel of cell lines using a custom pool of shRNAs. Several genes scored well across all cell lines. The top MEK inhibitor sensitizer was BRAF, thereby highlighting the importance of MAPK signaling in UM. Several non-MAPK pathway genes were also nominated and may represent novel pathways for the development of combination therapies.
Citation Format: Chelsea S. Place, Glenn S. Cowley, David E. Root, Levi A. Garraway. Synthetic lethal RNAi screens to nominate potential therapeutic combinations for uveal melanoma. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3429. doi:10.1158/1538-7445.AM2014-3429
Abstract only
Francisella tularensis ssp. tularensis
is a gram‐negative, facultative intracellular bacterium, which is classified as a Tier 1 Select Agent by the CDC due to its high infectivity and ...ease of propagation. Attempts to develop an effective vaccine have been unsuccessful, due in part to the organism's ability to suppress or bypass the host immune response early after infection.
F. tularensis
strains resistant to multiple antibiotics are a biowarfare threat. In the absence of an effective vaccine, new antibiotic targets and compounds are needed to ensure biodefense. Bacteria require at least one pathway to rescue ribosomes stalled at the end of mRNAs. The primary pathway for ribosome rescue is
trans
‐translation, which is conserved in >99% of sequenced bacterial genomes. Some bacterial species also have backup systems, such as ArfA or ArfB, which can rescue ribosomes in the absence of sufficient
trans
‐translation activity. Small molecule inhibitors of ribosome rescue have broad‐spectrum antimicrobial activity against bacteria grown in liquid culture. These compounds were tested against
F. tularensis
to determine if they can limit bacterial proliferation during infection of eukaryotic cells. The inhibitors KKL‐10 and KKL‐40 exhibited exceptional antimicrobial activity against both attenuated and fully virulent strains of
F. tularensis
in vitro and during ex vivo infection. Addition of KKL‐10 or KKL‐40 to macrophage or liver cells at any time after infection by
F. tularensis
prevented further bacterial proliferation. When macrophages were stimulated with the pro‐inflammatory cytokine interferon‐γ before being infected by
F. tularensis
, addition of KKL‐10 or KKL‐40 reduced intracellular bacteria by >99%, indicating that the combination of cytokine induced stress and a nonfunctional ribosome rescue pathway is fatal to
F. tularensis
. Neither KKL‐10 nor KKL‐40 were cytotoxic to eukaryotic cells in culture. These results demonstrate that ribosome rescue is required for
F. tularensis
growth at all stages of its infection cycle, and suggest that KKL‐10 and KKL‐40 will be good lead compounds for antibiotic development.
Support or Funding Information
This work was supported by NIH (AI077917 to Girish S. Kirimanjeswara and GM068720 to Kenneth C. Keiler) and CAS/PSU start up funds to Girish S. Kirimanjeswara. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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