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NLRP3 Inflammasome Plays a Redundant Role with Caspase 8 to Promote IL-1Β–Mediated Osteomyelitis

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NLRP3 Inflammasome Plays a Redundant Role with Caspase 8 to Promote IL-1Β–Mediated Osteomyelitis NLRP3 inflammasome plays a redundant role with caspase 8 to promote IL-1β–mediated osteomyelitis Prajwal Gurunga, Amanda Burtona, and Thirumala-Devi Kannegantia,1 aDepartment of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38105 Edited by Vishva M. Dixit, Genentech, San Francisco, CA, and approved March 9, 2016 (received for review February 1, 2016) Missense mutation in the proline-serine-threonine phosphatase- for CRMO are limited to the use of nonsteroidal antiinflammatory cmo interacting protein 2 (Pstpip2) gene results in the development of drugs (NSAIDs) and bisphosphonates (14). Pstpip2 mice have spontaneous chronic bone disease characterized by bone defor- proved to be a valuable tool in understanding the molecular mity and inflammation that is reminiscent of patients with chronic mechanisms involved in instigation of CRMO and bone diseases in cmo multifocal osteomyelitis (cmo). Interestingly, this disease is specif- general. Using Pstpip2 mice as a model to understand the eti- ically mediated by IL-1β but not IL-1α. The precise molecular path- ology of bone diseases, we previously demonstrated that IL-1R cmo ways that promote pathogenic IL-1β production in Pstpip2 mice signaling completely protected the progression of disease in these remain unidentified. Furthermore, how IL-1β provokes inflamma- mice (1). Adding to these studies, we and others showed that IL-1β, cmo tory bone disease in Pstpip2 mice is not known. Here, we dem- but not IL-1α, was important for induction of bone disease in these onstrate that double deficiency of Nod like receptor family, pyrin cmo mice (1, 15). More recent studies from our laboratory elucidated domain containing 3 (NLRP3) and caspase 8 in Pstpip2 mice a rather surprising redundant role for caspase 1 and caspase 8 provides similar protection as observed in caspase-1 and caspase- β cmo in the processing of IL-1 (2). The redundant roles of caspase 8–deficient Pstpip2 mice, demonstrating redundant roles for 1 and caspase 8 suggest that these caspases are functioning the NLRP3 inflammasome and caspase 8 in provoking osteomy- cmo independently in separate complexes to process the pathogenic elitic disease in Pstpip2 mice. Consistently, immunofluorescence cytokine IL-1β, unlike several other studies where caspase 1 studies exhibited distinct caspase-1 and caspase-8 puncta in dis- and caspase 8 have been proposed to be in the same complex Ptpn6spin eased neutrophils. Data from our chimera studies demon- (16). In this study, we sought to understand the upstream β strated that IL-1 produced by hematopoietic cells is sensed by the molecules that assemble the caspase-1 and caspase-8 complexes. radioresistant compartment to promote bone disease. Furthermore, Furthermore, we undertook an innovative chimeric approach to our results showed that the IL-1β signaling is unidirectional and β elucidate the IL-1R signaling feedback loop in hematopoietic and feedback signaling of IL-1 onto the hematopoietic compartment radioresistant compartments and determine how IL-1β provoked cmo is not important for disease induction. In conclusion, our studies inflammatory osteomyelitis in Pstpip2 mice. have uncovered the combined actions of the NLRP3 inflammasome We demonstrated that the caspase-1 complex is assembled by and caspase 8 leading to IL-1β maturation and the directionality of cmo NLRP3, whereas caspase 8 does not engage the TNF signaling IL-1β in driving disease in Pstpip2 mice. pathways, suggesting involvement of an alternate complex. Importantly, the NLRP3 inflammasome and caspase 8 play NLRP3 | inflammasome | caspase 8 | caspase 1 | PSTPIP2 redundant roles in IL-1β processing and inducing disease in cmo Pstpip2 mice. Activation of distinct caspase-1 and -8 com- utoinflammatory bone diseases that include osteoporosis, plexes within the neutrophils provoked excessive IL-1β production. APaget’s disease, arthritis, periodontal disease, and chronic recurrent multifocal osteomyelitis (CRMO) are a major health Significance burden to human health. The incidence of these diseases is projected to rise due to the increase in human life expectancy and changes in diet. IL-1 therapies have shown some promise in IL-1 is a pleiotropic cytokine involved in a myriad of auto- inflammatory disorders. In proline-serine-threonine phospha- alleviating disease symptoms; however, IL-1 therapies target Pstpip2cmo β both IL-1β and IL-1α, which have known distinct functions (1–3). tase-interacting protein 2 ( ) mice, IL-1 provokes Furthermore, the signaling pathways that underlie IL-1–mediated autoinflammatory osteomyelitis. Here, we demonstrated the redundant roles of Nod like receptor family, pyrin domain con- autoinflammation in these bone diseases are mostly unknown. taining 3 (NLRP3) inflammasome with caspase 8 in the process- Proline-serine-threonine phosphatase-interacting protein 2 cmo ing of IL-1β in Pstpip2 mice. Identification of redundancy (Pstpip2) gene is located on chromosome 18 in both mouse and between NLRP3 inflammasome and caspase 8 is fundamental in human. PSTPIP2 is a member of the Fes/CIP4 homology-bin/ our understanding of the inflammasomes and alternative modes amphiphysin/rvs (F-BAR) family of proteins also known as the of IL-1β regulation in osteomyelitic disease. Moreover, IL-1β pombe cdc15 homology family proteins (4). The F-bar domain signaling connects distinct compartments in promoting the dis- of PSTPIP2 has been known to interact with phosphatidyl- ease, identifying previously unidentified checkpoints that could inositol bisphosphate, whereas its C-terminal domain binds be targeted for therapeutic purposes in similar disease settings. protein tyrosine phosphatases of the PEST (a domain rich in β – Thus, our studies have unraveled the complex IL-1 regulatory proline, glutamic acid, serine, and threonine) family (5 7). network in vivo in a mouse model of osteomyelitis that will be Recent studies have shown that PSTPIP2 can also interact with useful for autoinflammatory diseases in general. inhibitory enzymes CsK and SHIP1 (8). Whereas human pa- PSTPIP2 tients with genetic mutations in the gene have not Author contributions: P.G. and T.-D.K. designed research; P.G. and A.B. performed re- been identified, missense L98P mutation in the gene Pstpip2 in search; P.G., A.B., and T.-D.K. analyzed data; and P.G. and T.-D.K. wrote the paper. mice results in severe autoinflammatory disease of the bones The authors declare no conflict of interest. that mimics CRMO in humans and thus these mice are re- This article is a PNAS Direct Submission. Pstpip2cmo – ferred to as mice (9 11). Patients with CRMO are 1To whom correspondence should be addressed. Email: thirumala-devi.kanneganti@ mostly composed of children and present with a wide range of stjude.org. symptoms ranging from bone inflammation, destruction, and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. deformity (12, 13). However, the current treatment options 1073/pnas.1601636113/-/DCSupplemental. 4452–4457 | PNAS | April 19, 2016 | vol. 113 | no. 16 www.pnas.org/cgi/doi/10.1073/pnas.1601636113 Downloaded by guest on September 27, 2021 cmo Finally, using bone marrow chimeras, we uncovered the complete bone disease in Pstpip2 mice, suggesting NLRP3 inflammasome IL-1β signaling loop, whereby IL-1β produced by neutrophils as the caspase-1 complex that plays a redundant role with caspase 8. (hematopoieitic compartments) is sensed by IL-1R on radio- Recent studies published from our laboratory and others have resistant cells to provoke autoinflammation and bone disease. shown that caspase 8 and caspase 1 can localize in the same inflammasome puncta containing ASC to process IL-1β (16–18). Results However, inflammasome-independent functions for caspase 8 in NLRP3 Inflammasome Plays a Redundant Role with Caspase 8 to Drive processing IL-1β have also been described (19–22). Our obser- Disease in Pstpip2cmo Mice. Whereas initial studies suggested that vation that deficiency of either caspase 1 or caspase 8 did not cmo NLRP3, Apoptosis-associated speck-like protein containing a prevent disease progression in Pstpip2 mice indicates that the card (ASC), and caspase 1 (inflammasome) were dispensable for two caspases are part of distinct and independently activated β– Pstpip2cmo complexes (Fig. 1 and ref. 2). Neutrophils are critical for disease IL-1 mediated disease in mice (1, 15), our recent cmo studies suggest that caspase 1 plays a redundant role with caspase induction in Pstpip2 mice, because the deletion of neutrophils cmo cmo 8 in promoting IL-1β secretion and disease in Pstpip2 mice can completely prevent disease in Pstpip2 mice (2). Thus, we (2). In line with these findings, we found no role for several asked whether caspase-1 and caspase-8 puncta could be visual- proteases (2) or cathepsin B and cathepsin G (Fig. S1). In- ized in neutrophils. Interestingly, we observed a significantly higher number of both caspase-1 and caspase-8 puncta in terestingly, deficiency of cathepsin C significantly delayed pro- cmo cmo Pstpip2 gression of disease in Pstpip2 mice, although most of the mice neutrophils compared with WT controls (Fig. 2). developed disease (Fig. S1). Whether cathepsin C plays a re- Whereas most of the neutrophils contained either caspase-1 or dundant role with other cathepsins or caspase 1 and caspase 8 is caspase-8 puncta alone, we also observed some cells that con- not known and will be investigated in the future. Our detailed tained both caspase-1 and caspase-8 puncta. Altogether, these data suggest that NLRP3 inflammasome plays a redundant role with a analysis of these mice showed that complete deficiency of both cmo cmo caspase-8 complex in promoting disease in Pstpip2 mice. caspase 1 and caspase 8 in Pstpip2 mice provides protection in more than 70% of the mice up to 120 d, although some mice TNF-Signaling Pathway Does Not Engage Inflammatory Caspase 8 in A B cmo demonstrate delayed clinical signs of disease (Fig. 1 and ). Pstpip2 Mice. One of the most well-established pathways that Interestingly, haploinsufficiency of caspase 8 or caspase 1 in re- lead to caspase-8 activation is the TNF-signaling pathway.
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