NLRP1-Dependent Pyroptosis Leads to Acute Lung Injury and Morbidity in Mice

Acute inflammation in response to both exogenous and endogenous danger signals can lead to the assembly of cytoplasmic inflammasomes that stimulate the activation of caspase-1. Subsequently, caspase-1 facilitates the maturation and release of cytokines and also, under some circumstances, the induction of cell death by pyroptosis. Using a mouse line lacking expression of NLRP1, we show that assembly of this inflammasome in cells is triggered by a toxin from Anthrax and that it initiates caspase-1 activation and release of IL-1β. Furthermore, NLRP1 inflammasome activation also leads to cell death, which escalates over three days following exposure to the toxin and culminates in acute lung injury and death of the mice. We show that these events are not dependent on production of IL-1β by the inflammasome but are dependent on caspase-1 expression. In contrast, MDP mediated inflammasome formation is not dependent on NLRP1, but NLRP3. Taken together, our findings show that assembly of the NLRP1 inflammasome is sufficient to initiate pyroptosis, which subsequently leads to a self-amplifying cascade of cell injury within the lung from which the lung cannot recover, eventually resulting in catastrophic consequences for the organism

Genetic loss of murine pyrin, the Familial Mediterranean Fever protein, increases interleukin-1β levels.

Familial Mediterranean Fever (FMF) is an inherited autoinflammatory disorder characterized by unprovoked episodes of fever and inflammation. The associated gene, MEFV (Mediterranean Fever), is expressed primarily by cells of myeloid lineage and encodes the protein pyrin/TRIM20/Marenostrin. The mechanism by which mutations in pyrin alter protein function to cause episodic inflammation is controversial. To address this question, we have generated a mouse line lacking the Mefv gene by removing a 21 kb fragment containing the entire Mefv locus. While the development of immune cell populations appears normal in these animals, we show enhanced interleukin (IL) 1β release by Mefv(-/-) macrophages in response to a spectrum of inflammatory stimuli, including stimuli dependent on IL-1β processing by the NLRP1b, NLRP3 and NLRC4 inflammasomes. Caspase-1 activity, however, did not change under identical conditions. These results are consistent with a model in which pyrin acts to limit the release of IL-1β generated by activation and assembly of inflammasomes in response to subclinical immune challenges

A loss of pyrin causes increased IL-1β protein levels in response to NLRP3 inflammasome stimuli.

<p>rpMΦs from <i>Mefv</i>^+/+ and <i>Mefv</i>^−/− littermate mice were exposed to LPS and the indicated stimuli as described in the method section. The concentration of IL-1β, <i>A</i>, and IL-6, <i>B</i>, cytokines in cell culture supernatants was determined by ELISA. <i>C</i>, LDH protein levels detected in cell supernatants. The total protein of cell lysates was measured to verify equivalent plating of cells. TiO2, titanium dioxide; MDP, muramyl dipeptide; Alum, aluminum hydroxide; CPPD, calcium pyrophosphate dihydrate; ATP, adenosine 5′ triphosphate. n = 6 mice per genotype. A student’s t test was used to calculate p values for WT versus <i>Mefv</i>^−/− cultures. #, p<0.05; *, p≤0.01 **, p≤0.001, ***, p<0.0001.</p

Caspase-1 (CASP-1) activity is not elevated in <i>Mefv^−/−</i> macrophages.

<p><i>A</i>, rpMΦs from <i>Mefv</i>^+/+ and <i>Mefv</i>^−/− littermate mice were exposed to LPS and ATP as indicated in the method section. Levels of caspase-1 (upper panel) and IL-1β (middle panel) were detected by western blot in cell lysates (lanes 1 and 2) and cell culture supernatant (lanes 3 and 4). An equivalent amount of total protein was loaded in each lane. The amount of protein in the lysate and supernatant was verified by staining with Ponceau S, major protein band is shown (lower panel). <i>B</i> and <i>C</i>, <i>Mefv</i>^+/+, <i>Mefv</i>^−/−, and <i>Casp1^−/−</i> mice were treated with 10 µg of lethal toxin (LT) or PBS vehicle alone by intratracheal instillation and bronchoalveolar lavage (BAL) was collected at 2 h after treatment. Cells from the BAL were stained with fluorescently labeled CASP-1 inhibitor (FLICA), FAM-YVAD-FMK. Cells were analyzed via flow cytometry. <i>B</i>, Data is expressed as the mean of the fluorescence intensity (MFI). <i>C</i>, A representative plot of the fluorescence intensity of LT or vehicle-treated cell populations. <i>D</i>, rpMΦs from <i>Mefv</i>^+/+ and <i>Mefv</i>^−/− littermate mice were treated with LPS and the indicated stimuli as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051105#pone-0051105-g003" target="_blank">Figures 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051105#pone-0051105-g004" target="_blank">4</a>. The concentration of IL-18 in cell culture supernatants was determined by ELISA. A student’s t test was used to calculate p values for <i>Mefv</i>^+/+ versus <i>Mefv</i>^−/− cultures. #, p<0.05; *, p<0.01.</p

Analysis of blood collected from <i>Mefv</i>^+/+ and <i>Mefv</i>^−/− mice.

<p>Blood was collected by cardiac puncture. Whole blood containing 5 mM EDTA was used for analysis on the Heska Hematology Analyzer by the Animal Clinical Chemistry and Gene Expression Laboratory at UNC-Chapel Hill. n = 13 and 13 mice. <b>WBC</b>- white blood cells, <b>RBC</b>- red blood cells, <b>PLT</b>- platelets, <b>LYMPH</b> – lymphocytes, <b>GRAN</b> – granulocytes, <b>MONO</b> – monocytes, <b>HCT</b> - hematocrit, <b>HGB</b> hemoglobin. Differences between Mefv^+/+ and Mefv^−/− were not statistically significant.</p

A loss of pyrin causes increased IL-1β release in response to NLRC4 and NLRP1b stimuli.

<p>rpMΦs from <i>Mefv</i>^+/+ and <i>Mefv</i>^−/− littermate mice were exposed to LPS and the indicated stimuli as indicated in the method section. The concentration of IL-1β, <i>A</i>, and IL-6, <i>B</i>, cytokines in cell culture supernatants was determined by ELISA. <i>C</i>, LDH protein levels detected in cell supernatants. The total protein of cell lysates was measured to verify equivalent plating of cells. LT, lethal toxin of <i>Bacillus anthracis</i>. n = 6 mice per genotype. Results are representative of at least 4 independent experiments. A student’s t test was used to calculate p values for WT versus <i>Mefv</i>^−/− cultures. #, p<0.05; *, p≤0.01.</p

Inflammatory response in <i>Mefv</i>^+/+ and <i>Mefv</i>^−/− mice after LPS treatment.

<p>The body weight lost was measured between 0 h and 20 h post-LPS treatment, immune cells present in the peritoneal lavage fluid, and MPO activity as a measure of neutrophil presence in lavage samples were evaluated 20 h post-LPS treatment. For body temperature changes, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051105#pone-0051105-g002" target="_blank">Figure 2F, G</a>. <b>n.a</b>. not analyzed. n = 18. Differences between Mefv^+/+ and Mefv^−/− mice within each dose of LPS treatment were not statistically significant.</p

NLRP1-Dependent Pyroptosis Leads to Acute Lung Injury and Morbidity in Mice

Acute inflammation in response to both exogenous and endogenous danger signals can lead to the assembly of cytoplasmic inflammasomes that stimulate the activation of caspase-1. Subsequently, caspase-1 facilitates the maturation and release of cytokines and also, under some circumstances, the induction of cell death by pyroptosis. Using a mouse line lacking expression of NLRP1, we show that assembly of this inflammasome in cells is triggered by a toxin from Anthrax and that it initiates caspase-1 activation and release of IL-1β. Furthermore, NLRP1 inflammasome activation also leads to cell death, which escalates over three days following exposure to the toxin and culminates in acute lung injury and death of the mice. We show that these events are not dependent on production of IL-1β by the inflammasome but are dependent on caspase-1 expression. In contrast, MDP mediated inflammasome formation is not dependent on NLRP1, but NLRP3. Taken together, our findings show that assembly of the NLRP1 inflammasome is sufficient to initiate pyroptosis, which subsequently leads to a self-amplifying cascade of cell injury within the lung from which the lung cannot recover, eventually resulting in catastrophic consequences for the organism

Genetic Loss of Murine Pyrin, the Familial Mediterranean Fever Protein, Increases Interleukin-1β Levels

Familial Mediterranean Fever (FMF) is an inherited autoinflammatory disorder characterized by unprovoked episodes of fever and inflammation. The associated gene, MEFV (Mediterranean Fever), is expressed primarily by cells of myeloid lineage and encodes the protein pyrin/TRIM20/Marenostrin. The mechanism by which mutations in pyrin alter protein function to cause episodic inflammation is controversial. To address this question, we have generated a mouse line lacking the Mefv gene by removing a 21 kb fragment containing the entire Mefv locus. While the development of immune cell populations appears normal in these animals, we show enhanced interleukin (IL) 1β release by Mefv (−/−) macrophages in response to a spectrum of inflammatory stimuli, including stimuli dependent on IL-1β processing by the NLRP1b, NLRP3 and NLRC4 inflammasomes. Caspase-1 activity, however, did not change under identical conditions. These results are consistent with a model in which pyrin acts to limit the release of IL-1β generated by activation and assembly of inflammasomes in response to subclinical immune challenges