Cryo-sensitive aggregation triggers NLRP3 inflammasome assembly in cryopyrin-associated periodic syndrome

  1. Tadayoshi Karasawa
  2. Takanori Komada
  3. Naoya Yamada
  4. Emi Aizawa
  5. Yoshiko Mizushina
  6. Sachiko Watanabe
  7. Chintogtokh Baatarjav
  8. Takayoshi Matsumura
  9. Masafumi Takahashi

  • Curated by eLife

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    Evaluation Summary:

    Karasawa and colleagues examine two gain-of-function mutations in NLRP3, which are known to cause cryopyrin-associated periodic syndromes (auto-inflammatory diseases with different manifestations), and demonstrate that both mutations appear to result in cryo-sensitive aggregated foci when expressed in cells. This is a very impactful and extensive body of work that is of broad interest to the fields of inflammasomes and autoinflammatory diseases. Data presented support the conclusions, and the findings are translational for patients and applicable to our general understanding of inflammasome function. However, specific issues raised by the Reviewers should be addressed.

    (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 #2 and Reviewer #3 agreed to share their name with the authors.)

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Abstract

Cryopyrin-associated periodic syndrome (CAPS) is an autoinflammatory syndrome caused by mutations of NLRP3 gene encoding cryopyrin. Familial cold autoinflammatory syndrome, the mildest form of CAPS, is characterized by cold-induced inflammation induced by the overproduction of IL-1β. However, the molecular mechanism of how mutated NLRP3 causes inflammasome activation in CAPS remains unclear. Here, we found that CAPS-associated NLRP3 mutants form cryo-sensitive aggregates that function as a scaffold for inflammasome activation. Cold exposure promoted inflammasome assembly and subsequent IL-1β release triggered by mutated NLRP3. While K + efflux was dispensable, Ca 2+ was necessary for mutated NLRP3-mediated inflammasome assembly. Notably, Ca 2+ influx was induced during mutated NLRP3-mediated inflammasome assembly. Furthermore, caspase-1 inhibition prevented Ca 2+ influx and inflammasome assembly induced by the mutated NLRP3, suggesting a feed-forward Ca 2+ influx loop triggered by mutated NLRP3. Thus, the mutated NLRP3 forms cryo-sensitive aggregates to promote inflammasome assembly distinct from canonical NLRP3 inflammasome activation.

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  1. Version published to 10.7554/elife.75166 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    Karasawa and colleagues report in this manuscript the aggregation of NLRP3 mutants associated with a group of autoinflammatory diseases called the cryopyrin-associated periodic syndromes (CAPS). Gain of function mutations in NLRP3 is associated with the systemic inflammatory characteristics in these diseases. This manuscript reports that CAPS-associated NLRP3 mutants (L353P and D303N) form cryo-sensitive aggregates, which function as scaffolds for NLRP3 inflammasome assembly in a NEK7- and potassium efflux-independent manner. Another key finding of this paper is the sensitivity of NLRP3 mutant aggregation to calcium. The strength of the manuscript is elegant immunofluorescence studies demonstrating the cold-sensitive aggregation of NLRP3 mutants. However, the role of calcium in NLRP3 CAPS mutant aggregation and inflammasome assembly needs clarification.

    We appreciate your insight into our study and helpful suggestion to improve our manuscript. As the reviewer pointed out, calcium is a critical regulator of aggregate formation by CAPS-associated NLRP3 mutants. In the revised manuscript, we further clarified the pivotal role of pannexin-1-mediated Ca2+ influx in inflammasome activation induced by mutated NLRP3. We have taken all of these comments and suggestions into account in the revision of our manuscript (changes are marked in red font in the revised manuscript). With respect to other specific comments, we performed several experiments and have responded as below.

    Reviewer #2 (Public Review):

    In this manuscript aggregation and activation of mutant NLRP3 at normal or low temperature is examined in several ways, which is a strength of the manuscript. In particular the imaging studies are performed in two cell lines, and appropriate quantification is usually provided. However, when considering the effect of temperature on the number of foci, some quantification on the area of the foci should also be considered, as the total amount of NLRP3 appears unchanged. Temperature could also have effects on pore formation and phagosomal rupture, so additional mechanisms of NLRP3 activation as control should also be considered. This manuscript also suggests that the effect of the two mutations in NLRP3 that are studied is independent of K+ efflux, MCC950 inhibition and NEK7, but dependent on Calcium influx. This appears reasonable but may require further controls. However I remain confused as to the importance for this as a feed-forward mechanism regulated by caspase-1 activation and this appears to contradict earlier data in the manuscript where the NLRP3 mutants formed foci independent of ASC.

    We appreciate your insight into our study and helpful suggestions. According to the suggestions, we performed additional experiments and have amended the manuscript. In particular, we validated the effect of caspase-1 inhibition on Ca2+ influx and subsequent inflammasome assembly using genetically deficient cells. Furthermore, our additional data suggest that pannexin 1 channel plays a pivotal role in a feed-forward mechanism of mutated NLRP3-mediated inflammasome assembly. We have taken all of these comments and suggestions into account in the revision of our manuscript (changes are marked in red font in the revised manuscript). With respect to other specific comments, we also performed several experiments and have responded as below.

    Reviewer #3 (Public Review):

    In their manuscript, "Cryo-sensitive aggregation triggers NLRP3 inflammasome assembly in cryopyrin-associated periodic syndrome,", Karasawa et al. use in vitro models to investigate the mechanism of CAPS associated mutations. They use confocal microscopy of several cell lines transfected with fluorescently tagged constructs, western blot, ELISA, qPCR, calcium and pyroptosis imaging, and inducible systems or inhibitors of NLRP3, caspase 1 and calcium signaling to investigate the mechanism of cold induction of NLRP3.

    We appreciate your positive comments and helpful suggestions that have helped us to improve our manuscript. In the revised manuscript, we further investigated cryo-sensitive aggregation of other CAPS-associated NLRP3 mutants. We believe that additional data support our conclusion. We have taken all of these comments and suggestions into account in the revision of our manuscript (changes are marked in red font in the revised manuscript). With respect to other specific comments, we performed several experiments.

  3. eLife

    Evaluation Summary:

    Karasawa and colleagues examine two gain-of-function mutations in NLRP3, which are known to cause cryopyrin-associated periodic syndromes (auto-inflammatory diseases with different manifestations), and demonstrate that both mutations appear to result in cryo-sensitive aggregated foci when expressed in cells. This is a very impactful and extensive body of work that is of broad interest to the fields of inflammasomes and autoinflammatory diseases. Data presented support the conclusions, and the findings are translational for patients and applicable to our general understanding of inflammasome function. However, specific issues raised by the Reviewers should be addressed.

    (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 #2 and Reviewer #3 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    Karasawa and colleagues report in this manuscript the aggregation of NLRP3 mutants associated with a group of auto-inflammatory diseases called the cryopyrin-associated periodic syndromes (CAPS). Gain of function mutations in NLRP3 is associated with the systemic inflammatory characteristics in these diseases. This manuscript reports that CAPS-associated NLRP3 mutants (L353P and D303N) form cryo-sensitive aggregates, which function as scaffolds for NLRP3 inflammasome assembly in a NEK7- and potassium efflux-independent manner. Another key finding of this paper is the sensitivity of NLRP3 mutant aggregation to calcium. The strength of the manuscript is elegant immunofluorescence studies demonstrating the cold-sensitive aggregation of NLRP3 mutants. However, the role of calcium in NLRP3 CAPS mutant aggregation and inflammasome assembly needs clarification.

  5. eLife

    Reviewer #2 (Public Review):

    In this manuscript aggregation and activation of mutant NLRP3 at normal or low temperature is examined in several ways, which is a strength of the manuscript. In particular the imaging studies are performed in two cell lines, and appropriate quantification is usually provided. However, when considering the effect of temperature on the number of foci, some quantification on the area of the foci should also be considered, as the total amount of NLRP3 appears unchanged. Temperature could also have effects on pore formation and phagosomal rupture, so additional mechanisms of NLRP3 activation as control should also be considered.

    This manuscript also suggests that the effect of the two mutations in NLRP3 that are studied is independent of K+ efflux, MCC950 inhibition and NEK7, but dependent on Calcium influx. This appears reasonable but may require further controls. However I remain confused as to the importance for this as a feed-forward mechanism regulated by caspase-1 activation and this appears to contradict earlier data in the manuscript where the NLRP3 mutants formed foci independent of ASC.

  6. eLife

    Reviewer #3 (Public Review):

    In their manuscript, "Cryo-sensitive aggregation triggers NLRP3 inflammasome assembly in cryopyrin-associated periodic syndrome,", Karasawa et al. use in vitro models to investigate the mechanism of CAPS associated mutations. They use confocal microscopy of several cell lines transfected with fluorescently tagged constructs, western blot, ELISA, qPCR, calcium and pyroptosis imaging, and inducible systems or inhibitors of NLRP3, caspase 1 and calcium signaling to investigate the mechanism of cold induction of NLRP3.

    The strengths of this paper are:
    1. Very carefully performed studies with novel findings using different cell lines, constructs, and techniques
    2. Data and interpretation that come together for an understandable story and a clear model

    The primary weaknesses are:
    1. Mechanistic data that does not necessarily agree with clinical findings

    Overall the authors achieved their aims of elucidating the mechanisms of CAPS mutation induced inflammation

    This work has impact on the treatment of patients and our understanding of NLRP3 inflammasome mechanisms

  7. Version published to 10.1101/2021.10.05.463273v2 on bioRxiv
  8. Version published to 10.1101/2021.10.05.463273v1 on bioRxiv