Abstract
Chronic recurrent multifocal osteomyelitis (CRMO) in humans can be modeled in Pstpip2cmo mice, which carry a missense mutation in the proline-serine-threonine phosphatase-interacting protein 2 (Pstpip2) gene. As cmo disease in mice, the experimental model analogous to human CRMO, is mediated specifically by IL-1β and not by IL-1α, delineating the molecular pathways contributing to pathogenic IL-1β production is crucial to developing targeted therapies. In particular, our earlier findings support redundant roles of NLR family pyrin domain-containing 3 (NLRP3) and caspase-1 with caspase-8 in instigating cmo However, the signaling components upstream of caspase-8 and pro-IL-1β cleavage in Pstpip2cmo mice are not well-understood. Therefore, here we investigated the signaling pathways in these mice and discovered a central role of a nonreceptor tyrosine kinase, spleen tyrosine kinase (SYK), in mediating osteomyelitis. Using several mutant mouse strains, immunoblotting, and microcomputed tomography, we demonstrate that absent in melanoma 2 (AIM2), receptor-interacting serine/ threonine protein kinase 3 (RIPK3), and caspase recruitment domain-containing protein 9 (CARD9) are each dispensable for osteomyelitis induction in Pstpip2cmo mice, whereas genetic deletion of Syk completely abrogates the disease phenotype. We further show that SYK centrally mediates signaling upstream of caspase-1 and caspase-8 activation and principally up-regulates NF-κB and IL-1β signaling in Pstpip2cmo mice, thereby inducing cmo These results provide a rationale for directly targeting SYK and its downstream signaling components in CRMO.
Highlights
Chronic recurrent multifocal osteomyelitis (CRMO) in humans can be modeled in Pstpip2cmo mice, which carry a missense mutation in the proline–serine–threonine phosphatase– interacting protein 2 (Pstpip2) gene
The NLR family pyrin domain-containing 3 (NLRP3) inflammasome plays a redundant role with caspase-8 to promote disease progression in Pstpip2cmo mice, indicating that NLRP3 is an upstream regulator of caspase-1 activation [12], but our understanding of the upstream regulation of caspase-8 activation remains incomplete
absent in melanoma 2 (AIM2) induces caspase-8 activation in caspase-1– deficient macrophages in the context of several bacterial infections, including Burkholderia [21], Francisella [22], and Legionella [23]. Given their established functions in caspase-8 activation under various conditions, we explored the roles of receptor-interacting serine/ threonine protein kinase 3 (RIPK3) and AIM2 in mediating caspase-8 activation in Pstpip2cmo mice by analyzing cmo disease progression in NLRP3- and RIPK3-deficient Pstpip2cmo mice (Pstpip2cmoNlrp3Ϫ/ϪRipk3Ϫ/Ϫ) and NLRP3- and AIM2-deficient Pstpip2cmo mice (Pstpip2cmoNlrp3Ϫ/ϪAim2Ϫ/Ϫ)
Summary
The NLRP3 inflammasome plays a redundant role with caspase-8 to promote disease progression in Pstpip2cmo mice, indicating that NLRP3 is an upstream regulator of caspase-1 activation [12], but our understanding of the upstream regulation of caspase-8 activation remains incomplete. Were rescued in Pstpip2cmoNlrp3Ϫ/ϪSykfl/flLysMcre and Pstpip2cmo Sykfl/flLysMcre mice (Fig. 2, A–C) Taken together, these data suggest that SYK functions upstream of both caspase-1 and caspase-8 in inducing cmo disease, that SYK is sufficient and necessary for cmo disease induction, and that NLRP3 and CARD9 are dispensable for cmo disease progression. Myeloidspecific deletion of SYK in Pstpip2cmo mice abolished the increased induction of pro-IL-1 in BMDMs upon LPS stimulation relative to Pstpip2cmo BMDMs without affecting the expression of caspase-1 and caspase-8 (Fig. 3B) These findings suggest a primary role for SYK in mediating pro-IL-1 production and cmo disease progression. We further noticed that SYK deficiency did not affect the expression of pro-IL-1, NLRP3, and ASC, all of which are crucial components of inflammasome signaling (Fig. 3E) These data suggest that SYK does not regulate the caspase-1 and caspase-8 activation mediated by the classical NLRP3 trigger. Our data indicate that SYK regulates NF-B signaling, but not inflammasome activation, for the induction of pro-IL-1 to mediate disease progression in Pstpip2cmo mice
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