Suppression of Ribosomal Function Triggers Innate Immune Signaling through Activation of the NLRP3 Inflammasome

Some inflammatory stimuli trigger activation of the NLRP3 inflammasome by inducing efflux of cellular potassium. Loss of cellular potassium is known to potently suppress protein synthesis, leading us to test whether the inhibition of protein synthesis itself serves as an activating signal for the NLRP3 inflammasome. Murine bone marrow-derived macrophages, either primed by LPS or unprimed, were exposed to a panel of inhibitors of ribosomal function: ricin, cycloheximide, puromycin, pactamycin, and anisomycin. Macrophages were also exposed to nigericin, ATP, monosodium urate (MSU), and poly I:C. Synthesis of pro-IL-ß and release of IL-1ß from cells in response to these agents was detected by immunoblotting and ELISA. Release of intracellular potassium was measured by mass spectrometry. Inhibition of translation by each of the tested translation inhibitors led to processing of IL-1ß, which was released from cells. Processing and release of IL-1ß was reduced or absent from cells deficient in NLRP3, ASC, or caspase-1, demonstrating the role of the NLRP3 inflammasome. Despite the inability of these inhibitors to trigger efflux of intracellular potassium, the addition of high extracellular potassium suppressed activation of the NLRP3 inflammasome. MSU and double-stranded RNA, which are known to activate the NLRP3 inflammasome, also substantially inhibited protein translation, supporting a close association between inhibition of translation and inflammasome activation. These data demonstrate that translational inhibition itself constitutes a heretofore-unrecognized mechanism underlying IL-1ß dependent inflammatory signaling and that other physical, chemical, or pathogen-associated agents that impair translation may lead to IL-1ß-dependent inflammation through activation of the NLRP3 inflammasome. For agents that inhibit translation through decreased cellular potassium, the application of high extracellular potassium restores protein translation and suppresses activation of the NLRP inflammasome. For agents that inhibit translation through mechanisms that do not involve loss of potassium, high extracellular potassium suppresses IL-1ß processing through a mechanism that remains undefined

Social Transfer of Pathogenic Fungus Promotes Active Immunisation in Ant Colonies

Social contact with fungus-exposed ants leads to pathogen transfer to healthy nest-mates, causing low-level infections. These micro-infections promote pathogen-specific immune gene expression and protective immunization of nest-mates

Inhibition of protein synthesis by dsRNA and inhibition of IL-1ß processing by MG-132.

<p>A) BMDM were treated with or without 4 h of LPS priming, as indicated. Cells were then rinsed in fresh medium and treated with either LipofectAMINE 2000 or LipofectAMINE 2000-poly I:C complex for 4 h, in the presence or absence of 30 µM MG-132, as indicated. Cell lysates (cell) or media (medium) samples were subjected to immunoblotting with the antibodies indicated. B) BMDM were treated with either LipofectAMINE 2000 alone or with LipofectAMINE 2000-dsRNA complex for the times indicated. Fifteen minutes before each time-point, 1 µCi of [³H]-leucine was added, and leucine incorporation was terminated by trichloroacetic acid. Each treatment was conducted in triplicate wells, and values are shown as mean ± S.D. Percent incorporation of [³H]-leucine at each point was calculated as the [³H]-leucine incorporated into cells exposed to LipofectAMINE 2000-dsRNA complex/[³H]-leucine incorporated into cells exposed to LipofectAMINE 2000 alone×100.</p

MSU crystals inhibit protein synthesis at concentrations that induce processing and release of IL-1ß.

<p>WT BMDM were primed with LPS for 4 h prior to exposure MSU or MPU at indicated concentrations. A) BMDM in triplicate wells were pulse-labeled in medium containing [³H]-leucine for 15 min prior to harvest at the indicated times and the amount of [³H]-leucine incorporation was measured. B) Cells were harvested 4 h after addition of indicated concentrations of MSU and MPU. Cell lysates (cell) and culture medium (medium) were examined by immunoblotting. P38 MAPK was loading control.</p

Effect of extracellular potassium on IL-1 processing and release.

<p>A) Bone marrow-derived macrophages were plated in triplicate wells in 12-well plates and primed with 50 ng/ml of LPS for 4 h. Cells were then incubated in medium containing 130 mM NaCl/5 mM KCl or 5 mM KCl/130 mM NaCl in the presence of absence of 0.01 µg/mL ricin, 25 µg/mL cycloheximide, 10 µg/mL, 10 µg/mL emetine, 75 µg/mL puromycin, 0.2 µg/mL pactamycin, 10 µg/mL anisomycin, 3.4 µM nigericin, or 5 mM ATP for 4 h. Medium was collected, and p17 IL-1 was determined by ELISA. B) Macrophages were plated, primed with LPS and incubated in medium containing 130 mM NaCl/5 mM KCl (MEM-Na) or 5 mM NaKCl/130 mM KCl (MEM-K) in the presence or absence 5 mM ATP or 3.4 µM nigericin for 4 h. Proteins were precipitated from the media with TCA and analyzed by Western blotting. C) Macrophages were plated, primed with LPS, and incubated in medium containing 130 mM NaCl/5 mM KCl (MEM-Na) or 5 mM NaKCl/130 mM KCl (MEM-K) in the presence or absence 10 ng/mL ricin, 10 µg/mL emetine, or 25 µg/mL cycloheximide for 4 h. Proteins were precipitated with TCA and analyzed by Western blotting.</p

Proteasome inhibitors block processing and release of IL-1ß.

<p>A) LPS-primed WT BMDM were incubated in control medium or medium containing 10 ng/ml ricin or 25 µg/ml cycloheximide for 4 h. MG-132 (30 µM) or Bortezimib (0.5 µM) was included as indicated. Secreted IL-1ß was measured by ELISA in triplicate wells. B) LPS-primed WT BMDM were incubated in the presence or absence of MG-132 for 4 hours, in the presence or absence of inhibitors of protein synthesis, as indicated. Cell lysates (cell) and culture medium (medium) were examined by immunoblotting. C) LPS-primed or unprimed WT BMDM were or exposed to MSU, MG-132, or both for 4 h, as indicated. Cell lysates (cell) and culture medium (medium) were examined by immunoblotting.</p

Data from: Social transfer of pathogenic fungus promotes active immunisation in ant colonies

Due to the omnipresent risk of epidemics, insect societies have evolved sophisticated disease defences at the individual and colony level. An intriguing yet little understood phenomenon is that social contact to pathogen-exposed individuals reduces susceptibility of previously naive nestmates to this pathogen. We tested whether such social immunisation in Lasius ants against the entomopathogenic fungus Metarhizium anisopliae is based on active upregulation of the immune system of nestmates following contact to an infectious individual or passive protection via transfer of immune effectors among group members—that is, active versus passive immunisation. We found no evidence for involvement of passive immunisation via transfer of antimicrobials among colony members. Instead, intensive allogrooming behaviour between naive and pathogen-exposed ants before fungal conidia firmly attached to their cuticle suggested passage of the pathogen from the exposed individuals to their nestmates. By tracing fluorescence-labelled conidia we indeed detected frequent pathogen transfer to the nestmates, where they caused low-level infections as revealed by growth of small numbers of fungal colony forming units from their dissected body content. These infections rarely led to death, but instead promoted an enhanced ability to inhibit fungal growth and an active upregulation of immune genes involved in antifungal defences (defensin and prophenoloxidase, PPO). Contrarily, there was no upregulation of the gene cathepsin L, which is associated with antibacterial and antiviral defences, and we found no increased antibacterial activity of nestmates of fungus-exposed ants. This indicates that social immunisation after fungal exposure is specific, similar to recent findings for individual-level immune priming in invertebrates. Epidemiological modeling further suggests that active social immunisation is adaptive, as it leads to faster elimination of the disease and lower death rates than passive immunisation. Interestingly, humans have also utilised the protective effect of low-level infections to fight smallpox by intentional transfer of low pathogen doses (“variolation” or “inoculation”)

Monandria to Syngenesia

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Konrad_etal_2012_Figure 5AB

Konrad_etal_2012_Figure 5A