Critical Role for Inflammasome-Independent IL-1Β Production in Osteomyelitis
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Critical role for inflammasome-independent IL-1β production in osteomyelitis John R. Lukensa, Jordan M. Grossa, Christopher Calabreseb, Yoichiro Iwakurac, Mohamed Lamkanfid,e, Peter Vogelf, and Thirumala-Devi Kannegantia,1 aDepartment of Immunology, bSmall Animal Imaging Core, and fAnimal Resources Center and the Veterinary Pathology Core, St. Jude Children’s Research Hospital, Memphis, TN 38105; cInstitute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan; and dDepartmentofBiochemistryand eDepartment of Medical Protein Research, Ghent University, B-9000 Ghent, Belgium Edited by Ruslan Medzhitov, Yale University School of Medicine, New Haven, CT, and approved December 4, 2013 (received for review October 3, 2013) The immune system plays an important role in the pathophysiology signal through the IL-1 receptor (IL-1R) to elicit potent proin- of many acute and chronic bone disorders, but the specific in- flammatory responses (10). IL-1β is generated in a biologically flammatory networks that regulate individual bone disorders remain inactive proform that requires protease-mediated cleavage to be to be elucidated. Here, we characterized the osteoimmunological secreted and elicit its proinflammatory functions. Caspase-1– underpinnings of osteolytic bone disease in Pstpip2cmo mice. These mediated cleavage of IL-1β following inflammasome complex mice carry a homozygous L98P missense mutation in the Pombe formation is the major mechanism responsible for secretion of β Cdc15 homology family phosphatase PSTPIP2 that is responsible bioactive IL-1 in many disease models (11). Inflammasome- β for the development of a persistent autoinflammatory disease re- independent sources of IL-1 have also been suggested to con- sembling chronic recurrent multifocal osteomyelitis in humans. We tribute to inflammatory disease pathogenesis; however, very little β is known about the molecular regulation of these pathogenic found that improper regulation of IL-1 production resulted in sec- α ondary induction of inflammatory cytokines, inflammatory cell infil- pathways (12, 13). IL-1 , on the other hand, does not require tration in the bone, and unremitting bone inflammation. Aberrant cleavage to induce its biological activity and is passively released following inflammatory forms of cell death (14, 15). Il1β expression precedes the development of osteolytic damage in cmo In this study, we sought to investigate the immunological young Pstpip2 mice, and genetic deletion of Il1r and Il1β, but not cmo factors that contribute to bone disease in Pstpip2 mice. We Il1α, IMMUNOLOGY rescued osteolytic bone disease in mutant mice. Intriguingly, show that osteomyelitic disease in these mice is characterized by caspase-1 and nucleotide-binding oligomerization domain (NOD)- uncontrolled and unremitting inflammatory cytokine production like receptor family, pyrin domain containing 3 activation in the and immune cell infiltration. Furthermore, we report a critical Pstpip2cmo inflammasome complex were dispensable for -mediated role for IL-1β that is derived independently of inflammasome bone disease. Thus, our findings establish a critical role for inflam- activation in osteolytic disease progression. masome-independent production of IL-1β in osteolytic bone dis- ease and identify PSTPIP2 as a negative regulator of caspase-1– Results β autonomous IL-1 production. Osteomyelitis Development in Pstpip2cmo Mice Is Associated with cmo Altered Immune Cell Composition. Pstpip2 mice develop bone osteoimmunology | interleukin-1 | autoinflammation nodules on their tails and paw inflammation that is characterized by severe erythema and hind paw deformities by 7–14 wk of age one diseases including osteoporosis, chronic recurrent mul- (Fig. 1A). Microcomputed tomography (micro-CT) scans of the ’ inflamed areas reveal extensive loss of bone density and struc- Btifocal osteomyelitis (CRMO), Paget s disease, arthritis, and cmo periodontal disease are a major burden to human health and can tural malformation in the feet and tails of Pstpip2 mice (Fig. 1 cause debilitating pain, physical impairments, and morbidity. Regulated crosstalk between cells of the skeletal and immune Significance systems is required to maintain normal bone integrity and ho- meostasis (1). It is now widely accepted that inflammatory immune The IL-1 cytokines, IL-1α and IL-1β, are proinflammatory cyto- cells contribute centrally to the induction and perpetuation of kines that are implicated in numerous inflammatory and au- various inherited and induced bone disorders (2, 3). However, the toimmune diseases. This study demonstrates that dysregulated roles of specific inflammatory pathways and immune cell networks immune responses centrally contribute to the pathogenesis of in disease pathogenesis remain to be fully characterized for most osteomyelitis and identifies a critical role for IL-1β in driving the – bone disorders. Recently, missense mutations in the proline ser- inflammatory cascade that provokes bone destruction. Cas- ine–threonine phosphatase interacting protein 2 (PSTPIP2) were pase-1 activation in the inflammasome complex is the most shown to cause a bone disease in mice that is characterized by well established mechanism for IL-1β secretion. Interestingly, osteomyelitis and bone deformity (4–6). In particular, the chronic inflammasome-independent sources of IL-1β were found to multifocal osteomyelitis (cmo) mouse that carries a homozygous cmo provoke inflammation and osteolytic bone disease. Our find- L98P missense mutation in PSTPIP2 (referred to as Pstpip2 ings establish a unique role for inflammasome-independent mice) develops a chronic autoinflammatory disease that resembles IL-1β in autoinflammatory bone disease and osteomyelitis, and CRMO in humans (7). CRMO is a painful genetic inflammatory identify proline–serine–threonine phosphatase interacting protein disorder that primarily affects children and is characterized by bone 2 as a negative regulator of inflammasome-autonomous IL-1β. inflammation, destruction, and deformity (8). The etiology of CRMO remains unknown, and treatment is currently limited to the Author contributions: J.R.L., C.C., M.L., and T.-D.K. designed research; J.R.L., J.M.G., C.C., prescription of nonsteroidal anti-inflammatory drugs (NSAIDs) and P.V. performed research; Y.I. contributed new reagents/analytic tools; J.R.L., J.M.G., and bisphosphonates. C.C., M.L., P.V., and T.-D.K. analyzed data; and J.R.L. and T.-D.K. wrote the paper. Excessive IL-1 production and signaling has recently been The authors declare no conflict of interest. identified to centrally contribute to a spectrum of autoinflam- This article is a PNAS Direct Submission. matory diseases (9). Recent data suggest that IL-1 can directly 1To whom correspondence should be addressed. E-mail: thirumala-devi.kanneganti@ influence bone homeostasis, and dysregulation of IL-1 has been stjude.org. found to contribute to bone disorders such as osteoarthritis (2). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. IL-1 exists in two distinct forms, IL-1α and IL-1β, both of which 1073/pnas.1318688111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1318688111 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 Metacarpal ABWT Pstpip2cmo WT Pstpip2cmo CWT Pstpip2cmo D cmo WT Pstpip2 Caudal Vertebrae WT Pstpip2cmo WT 5 22.66 +1.4% E FG10 104 cmo WT Pstpip2 2.5 *** 103 2.0 WT Pstpip2cmo 44.2 + 6 1.5 102 1.8% 0 1.0 N.S. 0.5 0102 103 104 105 Pstpip2cmo N.S. MHCII *** 105 57.6 +5.1% 0.10 *** 4 #popLN cells x 10 10 0.05 * 103 0.00 *** 102 18.0 + cells 3.8% Tregs + + cells 0 + Tcells 2 3 4 5 Neutrophils 010 10 10 10 MHCII CD4 CD11b CD4 Fig. 1. Mutation in Pstpip2 results in osteomyelitis and altered immune cell composition. (A) Spontaneous induction of paw inflammation and osteo- arthropathy (Top) and tail kinks (Bottom)inPstpip2cmo mice at 7–14 wk of age. Black arrows denote tail kinks. (B and C) Isosurface microcomputed to- mography (micro-CT) scans (B) and bone density heat maps (C) of WT and Pstpip2cmo mice. Blue arrows highlight areas of bone deformity, and white arrows denote sites of bone deposition. (D) Hind paw metacarpal bone and caudal vertebrae hematoxylin and eosin (H&E) sections from WT and Pstpip2cmo mice. (E–G) WT and diseased Pstpip2cmo mice were harvested at 10–12 wk of age. (E) Representative images of popliteal lymph nodes (popLN). (F) Number (mean ± SEM) of popLN cells. Data are representative of three independent experiments with at least five to six mice per group. Tregs, Foxp3-expressing regulatory T cells. (G) Expression of MHCII and CD4 by popLN cells. Numbers in the FACS plots represent the mean ± SEM. Data are representative of three independent experiments with at least five to six mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. B and C). Histological evaluation of the metacarpal bones and earlier report showing that osteomyelitic disease ensues in the the caudal vertebrae further demonstrated osteolytic inflammation absence of T cells in a separate Pstpip2 genetic mouse model lupo that was accompanied by hypertrophic osteoarthropathy and (Pstpip2 mice) (5). Collectively, these results suggest that cmo multifocal severe eosinophilic granulomatous inflammation osteolytic bone disease in Pstpip2 mice may primarily be (Fig. 1D). Moreover, the inflammatory bone lesions led to linked to dysregulated innate immune signaling. secondary s.c. inflammation and fibrosis of the surrounding soft tissue (Fig. 1D). Pstpip2cmo Mutation