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

Abstract

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.

Figure 1. Generation of Mefv ^−/− mice.

A, Schematic showing the endogenous Mefv locus, gene-targeting construct, and targeted locus after homologous recombination with the targeting construct. Upon homologous recombination, the entire coding region and 5 kb on either side of the Mefv gene was replaced with a selectable neomycin resistance (neo^r) gene. Exons are depicted as filled boxes. The genomic fragment used in Southern blot analysis is indicated as the 3′ probe. B, Southern blot was used to screen offspring generated by the intercross of Mefv ^+/− mice. The 3′ probe detects a BamHI restriction fragment that is diagnostic of proper integration of the targeting construct at the endogenous Mefv locus (upper panel). The deleted region (DR) probe corresponds to DNA in exon 2 and identifies a region deleted during homologous recombination (lower panel) C, Northern blot analysis of RNA from Mefv ^+/+ and Mefv ^−/− mice. A full-length Mefv cDNA probe was used to detect endogenous expression of Mefv in elicited peritoneal neutrophils. Several bands were detected in the WT sample, which is consistent with the presence of multiple splice variants of Mefv. The two most intense bands are shown. All bands are absent in Mefv ^−/− mice. A probe specific to β-actin confirms equal loading of the RNA samples.

Figure 2. Loss of Mefv does not affect the response to LPS.

A, Relative expression of Mefv in bone marrow-derived macrophages (BMMΦ), resting peritoneal macrophages (rpMΦ), and elicited peritoneal macrophages (pMΦ) from WT mice was detected by real-time PCR. n ≥3 mice per cell type. Data are expressed as relative expression normalized to Gapdh and BMMΦ. B-D, rpMΦ from Mefv ^+/+ and Mefv ^−/− littermate mice were untreated or treated with 1 µg/mL LPS for 24 h. B, Il1β and Il6 expression at the transcript level is expressed as fold induced by LPS treatment. n = 3 mice per genotype. C-E, Concentration of IL-6 and IL-1β in supernatants from LPS-treated rpMΦ cultures as determined by ELISA. n = 6 mice per genotype. E, The concentration of lactate dehydrogenase (LDH) released into cell supernatants was used as an indicator of cell death. WT and Mefv ^−/− mice were treated with 2.5 mg/kg (F, n = 8 WT and 5 Mefv ^−/− mice) or 0.25 mg/kg (G, n = 18 and 19) crude LPS by i.p. injection at time 0 h, as indicated by arrows. Rectal temperature at the indicated times is shown in F and G.

Figure 3. A loss of pyrin causes increased IL-1β protein levels in response to NLRP3 inflammasome stimuli.

rpMΦs from Mefv ^+/+ and Mefv ^−/− littermate mice were exposed to LPS and the indicated stimuli as described in the method section. The concentration of IL-1β, A, and IL-6, B, cytokines in cell culture supernatants was determined by ELISA. C, 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 Mefv ^−/− cultures. #, p<0.05; *, p≤0.01 **, p≤0.001, ***, p<0.0001.

Figure 4. A loss of pyrin causes increased IL-1β release in response to NLRC4 and NLRP1b stimuli.

rpMΦs from Mefv ^+/+ and Mefv ^−/− littermate mice were exposed to LPS and the indicated stimuli as indicated in the method section. The concentration of IL-1β, A, and IL-6, B, cytokines in cell culture supernatants was determined by ELISA. C, 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 Bacillus anthracis. 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 Mefv ^−/− cultures. #, p<0.05; *, p≤0.01.

Figure 5. Caspase-1 (CASP-1) activity is not elevated in Mefv^−/− macrophages.

A, rpMΦs from Mefv ^+/+ and Mefv ^−/− 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). B and C, Mefv ^+/+, Mefv ^−/−, and Casp1^−/− 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. B, Data is expressed as the mean of the fluorescence intensity (MFI). C, A representative plot of the fluorescence intensity of LT or vehicle-treated cell populations. D, rpMΦs from Mefv ^+/+ and Mefv ^−/− littermate mice were treated with LPS and the indicated stimuli as in Figures 3 and 4. 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 Mefv ^+/+ versus Mefv ^−/− cultures. #, p<0.05; *, p<0.01.