Genetic variant in 3’ untranslated region of the mouse pycard gene regulates inflammasome activity
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Curated by eLife
Evaluation Summary:
Genetic differences in outbred species such as humans and differences in the epigenomic structure form the basis of the large variability in the immune response. This work demonstrates that a single nucleotide change in the gene encoding for the universal inflammasome adaptor protein ASC regulates mRNA stability of Pycard and thereby inflammasome function. A particular strength of the work is that the authors managed to show, using genetic alterations, that a single SNP in the Pycard gene sequence (rs33183533) between AKR and DBA/2 mice causes variation in inflammasome activity. Given the relevance of inflammasome for various human pathologies, this work is important for a broad readership.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Quantitative trait locus mapping for interleukin-1β release after inflammasome priming and activation was performed on bone-marrow-derived macrophages (BMDM) from an AKRxDBA/2 mouse strain intercross. The strongest associated locus mapped very close to the Pycard gene on chromosome 7, which codes for the inflammasome adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC). The DBA/2 and AKR Pycard genes only differ at a single-nucleotide polymorphism (SNP) in their 3’ untranslated region (UTR). DBA/2 vs. AKR BMDM had increased levels of Pycard mRNA expression and ASC protein, and increased inflammasome speck formation, which was associated with increased Pycard mRNA stability without an increased transcription rate. CRISPR/Cas9 gene editing was performed on DBA/2 embryonic stem cells to change the Pycard 3’UTR SNP from the DBA/2 to the AKR allele. This single base change significantly reduced Pycard expression and inflammasome activity after cells were differentiated into macrophages due to reduced Pycard mRNA stability.
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Evaluation Summary:
Genetic differences in outbred species such as humans and differences in the epigenomic structure form the basis of the large variability in the immune response. This work demonstrates that a single nucleotide change in the gene encoding for the universal inflammasome adaptor protein ASC regulates mRNA stability of Pycard and thereby inflammasome function. A particular strength of the work is that the authors managed to show, using genetic alterations, that a single SNP in the Pycard gene sequence (rs33183533) between AKR and DBA/2 mice causes variation in inflammasome activity. Given the relevance of inflammasome for various human pathologies, this work is important for a broad readership.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive …
Evaluation Summary:
Genetic differences in outbred species such as humans and differences in the epigenomic structure form the basis of the large variability in the immune response. This work demonstrates that a single nucleotide change in the gene encoding for the universal inflammasome adaptor protein ASC regulates mRNA stability of Pycard and thereby inflammasome function. A particular strength of the work is that the authors managed to show, using genetic alterations, that a single SNP in the Pycard gene sequence (rs33183533) between AKR and DBA/2 mice causes variation in inflammasome activity. Given the relevance of inflammasome for various human pathologies, this work is important for a broad readership.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #2 Public Review:
In this manuscript, Ritchey and colleagues studied an intercross of two inbred mouse strains for their inflammasome response to interrogate the genetic basis for enhanced inflammasome activity. This was spurred by the observation that bone marrow-derived macrophages (BMDM) from DBA/2 mice showed an approximately 2-fold enhanced NLRP3 inflammasome response compared to BMDMs from AKR mice. To explore this phenomenon, they stimulated BMDMs from DBA/2 and AKR intercrosses (F4 generation) with NLRP3 agonists and then studied the ensuing IL-1β response. Conducting quantitative trait locus (QTL) mapping the authors then identified a region on chromosome 7 to have the highest LOD score for the phenotype studied (this region was named Irm3). The Irm3 region encompasses the 134.80-138.45 Mb interval on chromosome 7 that …
Reviewer #2 Public Review:
In this manuscript, Ritchey and colleagues studied an intercross of two inbred mouse strains for their inflammasome response to interrogate the genetic basis for enhanced inflammasome activity. This was spurred by the observation that bone marrow-derived macrophages (BMDM) from DBA/2 mice showed an approximately 2-fold enhanced NLRP3 inflammasome response compared to BMDMs from AKR mice. To explore this phenomenon, they stimulated BMDMs from DBA/2 and AKR intercrosses (F4 generation) with NLRP3 agonists and then studied the ensuing IL-1β response. Conducting quantitative trait locus (QTL) mapping the authors then identified a region on chromosome 7 to have the highest LOD score for the phenotype studied (this region was named Irm3). The Irm3 region encompasses the 134.80-138.45 Mb interval on chromosome 7 that encodes for 66 genes. Given its established role in inflammasome signaling and also a strong cis eQTL LOD score, the authors focused on Pycard in the following. Comparing the two mouse strains, the authors noted an SNV in the 3' UTR of the Pycard gene with differing genotypes for DBA/2 and AKR mice. This SNV is located just downstream the stop codon, a region that seems to display little conservation across different mammalian species. Comparing ASC protein expression, the authors noted increased levels of ASC in BMDMs from DBA/2 mice, a finding that also translated into higher amounts of ASC speck levels following inflammasome stimulation. Subsequent experiments indicated that Pycard mRNA levels of BMDMs from DBA/2 mice displayed a longer half-life, while Pycard mRNA transcription or splicing was not affected. Modeling the 3' UTR region of interest furthermore suggested that the SNV impacts on the structure of this region. To validate the causal role of this SNV in regulating Pycard expression, the authors generated DBA/2 ES cells, in which they changed the genotype of this SNV into the corresponding AKR variant. Comparing ES-cell-derived macrophages of the parental DBA/2 genotype to the AKR-adapted Pycard genotype, the authors found that ASC expression levels were indeed decreased and that this reduced expression translated into a reduced NLRP3 inflammasome response in these cells. Altogether, these data suggest that an SNV in the 3' UTR of the murine Pycard gene impacts the stability of its mRNA, which translates into altered ASC protein levels and thereby the activity of inflammasome pathways.
Strength:
The conclusions of this paper are well supported by data and there are no major gaps or flaws in the line of reasoning. A particular stronghold is the functional validation of the here-identified SNV using a CRISPR-based point mutagenesis approach. This set of data provides a high level of confidence for the proposed model.
Weakness:
While this manuscript provides an elegant QTL mapping approach to identify differential expression of Pycard as a major regulator of inflammasome activity in murine BMDMs, the outcome of this study does not provide any new biological insight into inflammasome biology. The fact that differential expression of ASC impacts on inflammasome activity is well expected based on its firmly established role in inflammasome signaling.
Unfortunately, the here-identified mechanism of the differential regulation of the half-life of the Pycard mRNA is not conserved in other species, which precludes any extrapolations to other organisms. Moreover, as also correctly summarized by the authors, there is currently no evidence that genetic variants leading to differential ASC expression in humans would impact on human health or disease. These shortcomings obviously limit the conceptual advance and relevance of the here-identified mechanism. -
Reviewer #1 Public Review:
Genetic differences in outbred species such as humans and differences in the epigenomic structure form the basis of the large variability in the immune response. In particular, the inflammasome is highly regulated at multiple levels, including the post-transcriptional and post-translational levels. Inflammasome responses towards a myriad of triggers are associated with disease development in murine models of disease. Furthermore, clinical trials are ongoing testing the ability of inflammasome inhibitory small molecules to prevent or ameliorate inflammasome-driven pathologies in patient populations.
This manuscript identified that a single nucleotide change in the gene encoding for the universal inflammasome adaptor protein ASC regulates mRNA stability of Pycard and thereby inflammasome function. A particular …
Reviewer #1 Public Review:
Genetic differences in outbred species such as humans and differences in the epigenomic structure form the basis of the large variability in the immune response. In particular, the inflammasome is highly regulated at multiple levels, including the post-transcriptional and post-translational levels. Inflammasome responses towards a myriad of triggers are associated with disease development in murine models of disease. Furthermore, clinical trials are ongoing testing the ability of inflammasome inhibitory small molecules to prevent or ameliorate inflammasome-driven pathologies in patient populations.
This manuscript identified that a single nucleotide change in the gene encoding for the universal inflammasome adaptor protein ASC regulates mRNA stability of Pycard and thereby inflammasome function. A particular strength of this manuscript is that the authors managed to show, using genetic alterations, that the single SNP in the Pycard gene sequence (rs33183533) between AKR and DBA/2 mice is the cause of variance in inflammasome activity. Given the relevance of inflammasome for various human pathologies, this work is important for a broad readership.
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