Hemorrhagic Shock Primes For NLRP3Inflammasome Activation In The Lung Through Impaired Pyrin Induction

Hemorrhagic shock (HS) promotes the development of systemic inflammatoryresponse syndrome (SIRS) and organ injury by activating and priming the innate immunesystem for an exaggerated inflammatory response through, as of yet, unclear mechanisms.IL-1also plays an important role in the development of post-HS SIRS and active IL-1production is tightly controlled by the inflammasome. Pyrin, a protein of781amino-acidswith Pyrin domain (PYD) at the N-terminal, negatively regulates inflammasome activationthrough interaction with nucleotide-binding oligomerization domain-like receptor protein(NLRP). Expression of Pyrin can be induced by LPS and cytokines, and IL-10is a knownpotent inducer of Pyrin expression in macrophages. In the present study, we tested thehypothesis that HS downregulates IL-10, and therefore decreases Pyrin expression topromote inflammasome activation and subsequent IL-1processing and secretion in thelungs. Our results show that LPS, while activating NLRP3inflammasome in the lungs,also induced Pyrin expression, which in turn suppressed inflammasome activation. Moreimportantly, LPS-mediated upregulation of IL-10enhanced Pyrin expression, which serves,particularly in later phases, as a potent negative feedback mechanism regulatinginflammasome activation. However, HS-mediated suppression of IL-10expression inalveolar macrophages (AM) attenuated the upregulation of Pyrin in AM and lungendothelial cells, and thereby significantly enhanced inflammasome activation and IL-1secretion in the lungs. This study demonstrates a novel mechanism by which HSsuppresses negative feedback regulation of NLRP3inflammasome to enhance IL-1secretion in response to subsequent LPS challenge, and so primes for inflammation. PART1: HS augments Nlrp3inflammasome activation in response to LPS in the lung.1HS augments Nlrp3inflammasome activation in response to LPS in the lung;To determine whether enhanced activation of inflammasome might contribute to shock-enhanced IL-1processing, we examined inflammasome activation in the lung bydetecting the association of Nlrp3and ASC, as well as caspase-1cleavage in animalstreated with LPS with or without prior HS. Lung tissue from sham and shock alone animalsdemonstrated a very low level of association between Nlrp3and ASC, as well as anundetectable caspase-1cleavage. Administration of LPS to sham animals induced anincrease in the association betweenNlrp3and ASC and cleavage of caspase-1in the lung by4h, which increased further by8h. Animals subjected to HS before LPS exhibited at8h a noticeable increase in theassociationbetween Nlrp3and ASC and cleavage of caspase-1as compared with that in the lungsfrom sham/LPS-treated animals at the same time point. The increased activation ofinflammasome and caspase-1seen in the lungs from HS/LPS-treated animals resulted in amarked increase in IL-1in BAL fluid, which represents the secretion of IL-1frompulmonary cells.2HS primes for enhanced Nlrp3inflammasome activation and IL-1secretion inthe AM.To determine whether AM contribute to the enhanced inflammasome activation detectedin whole lung tissue, BAL cells were recovered at2h after HS/resuscitation and enrichedfor AM by using immunomagnetic separation system. The AM were then incubated invitro in the presence or absence of LPS (1mg/ml) to evaluate activation of inflammasomeand caspase-1, as well as IL-1release. AM from sham animals treated with LPS exhibited an increased association of Nlrp3and ASC and increased caspase-1cleavage at4and8h. There was also increased IL-1secretion in the cell-culture medium by8h.However, in the AM that were isolated from HS mice, LPS induced a markedly augmentedassociation of Nlrp3and ASC, cleavage of caspase-1, and release of IL-1.PART2: The mechanism of NLRP3inflammasome activation in the lung throughimpaired Pyrin induction post-hemorrhagic shock.1HS causes impaired upregulation of Pyrin in the lung;Pyrin has been suggested as an inhibitor of caspase-1activation and subsequentprocessing of IL-1. To address whether Pyrin is involved in the regulation ofinflammasome activation inthe lung in a setting of HS, we first detected the level of Pyrin expression in the lung andAM following HS and/or LPS. Using the animal model of HS/LPS treatments, we foundthat antecedent HS significantly impaired the expression of Pyrin protein in the lungs inresponse to LPS. This observation was recapitulated in the AM that were isolated fromeither sham or HS mice and treated with LPS ex vivo. LPS upregulated the expression ofPyrin protein in AM fromsham animals, whereas HS markedly attenuated the LPS-induced expression of Pyrin.2HS-suppressed IL-10expression is responsible for impaired Pyrin induction;We have previously reported that HS impairs LPS-induced upregulation of IL-10, whichin turn exaggerates lung inflammation. To elucidate whether IL-10expression impaired byHS contributes to reduced Pyrin induction, rIL-10(10mg/kg B.W.) or neutralizing Ab against IL-10(2mg/kg B.W.) was administered i.t. along with LPS or saline to sham or HSanimals at2h afterresuscitation, and Pyrin protein expression in the lungs was then detected at2and8hafterward. At the2-h time point, exogenous IL-10induced a greater increase in pulmonaryPyrin in both sham and HS mice as compared with that in the groups treated with LPSalone, and the combination of LPS and IL-10induced an even higher expression of Pyrinin the lungs. At8h after LPS, exogenous IL-10significantly enhanced the LPS-inducedPyrin expression in HS animals, and this was significantly attenuated in both sham and HSmice by neutralizing Ab against IL-10. These findings suggest that the failed upregulationof IL-10contributes to the suppressed Pyrin expression following HS/LPS, especially atthe later time point.3Impaired Pyrin induction contributes to HS-augmented inflammasome activation.Finally, we tested the hypothesis that HS impaired IL-10expression, and therefore,decreased Pyrin expression in the lungs is responsible for augmented inflammasomeactivation and IL-1secretion following HS/LPS. The results demonstrate an important role of IL-10and Pyrinin regulating IL-1secretion, predominantly through modulating activation of Nlrp3inflammasome. Taken together, we could draw these conclusions:1HS augments Nlrp3inflammasome activation in response to LPS in the lung;2HS primes for enhanced Nlrp3inflammasome activation and IL-1secretion in the AM.3HS causes impaired upregulation of Pyrin in the lung;4HS HS-suppressed IL-10expression is responsible for impaired Pyrin induction;5Impaired Pyrin induction contributes to HS-augmented inflammasome activation.