Pathways of innate immune sensing of DNA.

<p>(A) Cytosolic DNA from invading viruses and bacteria engage and activate AIM2 binding to the adaptor ASC. ASC mediates caspase-1-dependent pro-IL-1β/pro-IL-18 cleavage and secretion of their bioactive forms, IL-1β and IL-18. IL-1β and IL-18 are significant mediators of inflammatory responses to infection. (B) Four known cytosolic sensors are represented here. Lrrfip1 recognized viral DNA as well as RNA to induce IFNβ via a β-catenin-IRF3 transactivator pathway independently of the kinase TBK1. DAI can bind double-stranded B-form and atypical Z-form DNA to induce TBK1-IRF3-dependent IFNβ production. Evidence for the role of adaptors MAVS/STING in these pathways is lacking. IFI16 can directly bind viral DNA via its HIN200 domains and initiate IFNβ induction in a STING-TBK1- and IRF3-dependent manner. RNA polymerase III (Pol III) generates 5′ tri-phosphate RNA that is a ligand for RIG-I. RIG-I signals via the adaptor MAVS, subsequently activating ubiquitin ligase TRAF3 and subsequently TBK1 and IRF3. The ubiquitin binding protein RNF5 inhibits STING activation by targeting it to the proteasome, while TREX1 inhibits/prevents IFNβ production by degrading DNA substrate. (C) The receptor for advanced glycated end products (RAGE) and HMGB1 can bind extracellular CpG-rich DNA and transport it to a TLR9-positive compartment. Here, it is recognized by TLR9 and signals via MyD88 and the IKK kinase, IKKα, and IRF7 in pDCs to induce IFNα production. The cytosolic DExD/H box helicases DHX9/DHX36 can recognize cytosolic CpG DNA and initiate signaling to IRF7 via MyD88.</p

Malaria-induced NLRP12/NLRP3-dependent caspase-1 activation mediates IL-1β and hypersensitivity to bacterial superinfection.

<p>Step 1 –Phagocytes internalize <i>Plasmodium</i> DNA bound to hemozoin that activates TLR9 and the adaptor molecule named MyD88. <b>Step 2 –</b> MyD88 signaling triggers the expression of IL-12, which will initiate the production of IFN-γ by T lymphocytes and NK cells. <b>Step 3 –</b> Low levels of caspase-1-independent IL-1β induced by malaria infection. <b>Step 4 –</b> IFN-γ priming and MyD88 signaling in phagocytes will lead to enhanced expression of pro-caspase-1. K⁺ efflux as well as rupture (by hemozoin crystals) and release of lysosome contents will induce the assembly of ASC, NLRP3 and NLR12 inflammasomes and promote cleavage of pro-caspase-1. <b>Step 5 -</b> Bacterial superinfection triggers expression of high pro-IL-1β levels, in a TNF-α-dependent manner. Pro-IL-1β will be cleaved by active caspase-1 generated on <b>step 4</b>. Upon secondary bacterial infection, the malaria-primed macrophages will release deleterious amounts of IL-1β.</p

Treatment with IL-1RA prevents lethality in mice infected with <i>P. chabaudi</i> and challenged with a secondary bacterial infection.

<p>(<b>A</b>) At 7 days post-infection, mice were challenged with 10 µg of LPS and serum samples collected 9 hours later for cytokine measurements. The numbers within parenthesis indicate the percentage of lethality 24 hours after low dose (10 µg/mouse) LPS challenge. (<b>B</b>) Splenic macrophages (CD11b⁺F4/80⁺) and DCs (CD11c⁺MHC-II⁺) from mice at 7 days post-infection were stained with FLICA reagent in order to detect active caspase-1. (<b>C</b>) At day 7 post-infection the mice were treated with IL-1RA (anakinra) immediately prior to LPS challenge. Lethality was assessed from 12 to 48 hours post-LPS challenge. (<b>D</b>) At 7 days post-infection with <i>P. chabaudi</i>, sub-lethal sepsis was induced by CLP. A group of mice received treatment with IL-1RA (100 mg/kg/day) beginning 24 hours before the CLP procedure. Levels of circulating IL-1β were measured 24 hs after CLP. (<b>E</b>) Mice received peroral challenge with 10⁸ of <i>Salmonella typhimurium</i> at 5 days post-infection with <i>P. chabaudi.</i> A group of mice received treatment with IL-1RA (100 mg/kg/day) beginning 48 hours after bacterial challenge. The levels of circulating IL-1β were measured at 3 days post-<i>Salmonella</i> challenge. (<b>F</b>) Translocation of aerobic bacteria was quantified 24 hours after the CLP procedure. (<b>G</b>) Translocation of <i>S. typhimurium</i> was quantified 3 days after peroral challenge. We used 5 to 8 mice per group and results shown are representative of 2 independent experiments. Significant differences are *<i>p<0.01</i>, <i>**p<0.005 ***p<0.001</i> obtained in a Chi-square test.</p

Association of circulating IL-1β levels and high susceptibility to endotoxic shock in mice primed by infection with <i>P.chabaudi</i>.

<p>IL-1β release and high susceptibility to endotoxic shock. Mice were infected with 10⁵ parasitized red blood cells (i.p). At 7 days post-infection, a low dose LPS (10 µg/mouse) was inoculated and lethality evaluated 24 hours later. Parasitemia was defined by smears giemsa- stained. FLICA reagent was used to assess active caspase-1 in total splenocytes from mice at 7 days post-infection. The data were collected by flow cytometry and median fluorescence intensity (MFI) analyses performed using Flowjo software. The range of detection of circulating IL-1β was 15.6–1000 pg/ml and determined using and ELISA Duoset kit from R&D Systems.</p

NLRP3/NLRP12 containing inflammasomes and caspase-1 activation in PBMCs from <i>P. vivax</i> malaria patients.

<p>(<b>A</b>) PBMCs derived from <i>P. vivax</i> malaria patients and healthy donors were lysed, cross-linked by treatment with disuccinimidyl suberate <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003885#ppat.1003885-Moulds1" target="_blank">[70]</a>, and ASC oligomerization assessed by Western blot analysis. PBMCs from a healthy donor stimulated with LPS and nigericin were used as positive control. . (<b>B</b>) NLRP3, NLRP12 and AIM2 containing inflammasomes (specks) in monocytes from <i>P. vivax</i> malaria patients were visualized in a confocal microscope. (<b>C</b>) The bar graphs show the frequency of specks in monocytes derived from <i>P. vivax</i> malaria patients. We saw no specks on cells from healthy donors or cells from malaria patients incubated with the secondary antibody only. See also <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003885#ppat.1003885.s006" target="_blank">Figure S6</a></b>.</p

NLRP3/NLRP12-dependent activation of caspase-1 and pyroptosis in mice infected with <i>P. chabaudi</i>.

<p>(<b>A</b>) At 9 days post-infection, splenocytes from C57BL/6, and P2X7R^−/− mice were lysed and analyzed by Western blot employing caspase-1-specific antibody. (<b>B</b>) At 7 days post-infection, active caspase-1 by FLICA reagent, membrane integrity by propidium iodide, and cell size change by shift on FSC axis were assessed in splenic macrophages (CD11b⁺F4/80⁺) and DCs (CD11c⁺MHC-II⁺). (<b>C</b>) Splenocytes lysates from mice at 7 days post-infection were used to detected active caspase-1 by Western blot. A faint band of similar molecular weight of active caspase-1 that corresponds to IgG light chain is seen in the uninfected controls or in various infected knockout mice. The results presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003885#ppat-1003885-g003" target="_blank">figures 3A, 3B and 3C</a> are representative of 2 experiments that yield similar results. (<b>D</b>) A LPS dose of 10 µg/mouse was given intravenously at 7 days post-infection with <i>P.chabaudi</i> and sera collected 9 hours later, for measuring IL-1β and TNF-α levels. The numbers within parenthesis indicate the percentage of lethality 48 hours after LPS challenge (10 µg/mouse). The levels of IL-1β measured in the sera of infected C57BL/6, NLRP3^−/−, NLRP12, ASC^−/− and Casp-1^−/− were not different. The results are means + SEM of 10 mice from 2 independent experiments. Significant differences are indicated by **<i>p<0.001</i> obtained in the Mann-Whitney test.</p

Monocytes are the major source of active caspase-1 during malaria.

<p>PBMCs were obtained from either <i>P. vivax</i> or <i>P. falciparum</i> malaria patients as well as from healthy donors. (<b>A</b>) The gate was set based on monocytes profile in PBMCs from <i>P. vivax</i> infected patients. PBMCs were stained with anti-CD14 and anti-CD16 antibodies to determine the presence of different monocyte subsets. These cells were gated based on FSC and SSC to avoid neutrophil contamination. When the CD14^dimCD16⁺ gate was moved down in PBMCs from healthy donors or from malaria patients after treatment, we did not detect any active caspase-1. The bar graphs show the flow cytometry analysis of PBMCs from five <i>P. vivax</i> infected subjects before and after malaria treatment primaquine and chloroquine, as well as eight healthy donors. To determine the median fluorescence intensity (MFI) and frequency of (CD14⁺CD16⁻) and (CD14^dimCD16⁺) that are active caspase-1, we used the FLICA assay. (<b>B – top panel</b>) Active caspase-1 (p10) was detected in lysates of PBMCs from <i>P. vivax</i> or (<b>C – top panel</b>) <i>P. falciparum</i> infected individuals by Western blot. (<b>B and C – bottom panel</b>) PBMCs were stimulated with LPS (100 ng/ml) for 24 hours, and levels of IL-1β assessed in the culture supernatants by ELISA. Significant differences are *<i>p</i><<i>0.05</i> and **<i>p<0.005</i> as indicated by the unpaired <i>t</i> test with Welch correction or Mann-Whitney test when a normality test failed.</p

Caspase-1 activation, IL-1β production and pyroptosis in splenic macrophages and DCs from <i>P. chabaudi</i> infected mice.

<p>(<b>A</b>) Gene expression was determined in splenocytes of 3 C57BL/6 or MyD88^−/− mice at 6 days post-infection over 3 non-infected controls by Microarray analysis. (<b>B and F</b>) Splenocytes from C57BL/6, ASC^−/−, Casp-1^−/− or MyD88^−/− mice were stained and analyzed by FACs to gate macrophages (CD11b⁺F4/80⁺) and DCs (CD11c⁺MHC-II⁺). Active caspase-1 was evaluated by FLICA reagent, membrane integrity by nuclei staining with 7AAD, and cell size change by shift on FSC axis. The results are representative from 3 experiments that yield similar results. (<b>C</b>) On day 7 post-infection splenocytes from C57BL/6, ASC^−/−, Casp-1^−/− and MyD88^−/− mice were lysed by RIPA buffer and analyzed by Western blot employing an anti-caspase-1 antibody. A faint band of similar molecular weight of active caspase-1 corresponds to IgG light chain is seen in the infected ASC^−/− and Casp-1^−/− mice. (<b>D and G</b>) At 7 days post-infection mice were inoculated intravenously with 10 µg of LPS per mouse, and 9 hours later, sera was collected for measuring the levels of circulating IL-1β. The average levels of IL-1β in control and infected mice, before LPS challenge, were 64.2 and 434.2 pg/ml in figure D, and 82.2 and 602.4 pg/ml in figure G. These results are the means + SEM of 10–15 animals from 3 independent experiments that yield similar results. (<b>E</b>) CD11c⁺ and CD11b⁺ cells highly purified from spleens of C57BL/6 mice at day post-infection were cultured with LPS (1 µg/ml) and supernatants harvested 18 h later to measure the levels of IL-1β. As positive control we used the purified cells stimulated with LPS at same concentration followed by nigericin at 5 µM. Significant differences are indicated by *<i>p</i><0.01, **<i>p<0.001</i> and ***<i>p<0.0005</i> obtained in the Mann-Whitney test.</p