Critical role for inflammasome-independent IL-1β production in osteomyelitis

Abstract

The immune system plays an important role in the pathophysiology of many acute and chronic bone disorders, but the specific inflammatory networks that regulate individual bone disorders remain to be elucidated. Here, we characterized the osteoimmunological underpinnings of osteolytic bone disease in Pstpip2(cmo) mice. These mice carry a homozygous L98P missense mutation in the Pombe Cdc15 homology family phosphatase PSTPIP2 that is responsible for the development of a persistent autoinflammatory disease resembling chronic recurrent multifocal osteomyelitis in humans. We found that improper regulation of IL-1β production resulted in secondary induction of inflammatory cytokines, inflammatory cell infiltration in the bone, and unremitting bone inflammation. Aberrant Il1β expression precedes the development of osteolytic damage in young Pstpip2(cmo) mice, and genetic deletion of Il1r and Il1β, but not Il1α, rescued osteolytic bone disease in mutant mice. Intriguingly, caspase-1 and nucleotide-binding oligomerization domain (NOD)-like receptor family, pyrin domain containing 3 activation in the inflammasome complex were dispensable for Pstpip2(cmo)-mediated bone disease. Thus, our findings establish a critical role for inflammasome-independent production of IL-1β in osteolytic bone disease and identify PSTPIP2 as a negative regulator of caspase-1-autonomous IL-1β production.

Keywords: autoinflammation; interleukin-1; osteoimmunology.

Fig. 1.

Mutation in Pstpip2 results in osteomyelitis and altered immune cell composition. (A) Spontaneous induction of paw inflammation and osteoarthropathy (Top) and tail kinks (Bottom) in Pstpip2^cmo mice at 7–14 wk of age. Black arrows denote tail kinks. (B and C) Isosurface microcomputed tomography (micro-CT) scans (B) and bone density heat maps (C) of WT and Pstpip2^cmo 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 Pstpip2^cmo mice. (E–G) WT and diseased Pstpip2^cmo 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.

Fig. 2.

Dysregulated IL-1 signaling centrally contributes to inflammatory bone disease in Pstpip2^cmo mice. (A) Levels of cytokines in the hind paws of WT and diseased Pstpip2^cmo mice. Each point represents an individual mouse, and the line represents the mean ± SEM. Data are representative of three independent experiments with at least eight mice per group. (B and C) Il1β and Tnfα mRNA expression in the hind paws of WT, diseased Pstpip2^cmo (B), and asymptomatic, young Pstpip2^cmo (4–6 wk of age) mice (C). Data are representative of three independent experiments with at least four mice per group. (D) Incidence of disease in WT, Pstpip2^cmo, and Pstpip2^cmoxIl1r^−/− mice over time. (E–G) Representative paw images (E) and isosurface micro-CT tail (F) and paw (G) scans from WT, Pstpip2^cmo, and Pstpip2^cmoxIl1r^−/− mice. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 3.

Deletion of IL-1β prevents Pstpip2^cmo-mediated bone disease. (A and B) Representative paw images (A) and tail CT scans (B) from WT and Pstpip2^cmo mice that were crossed with mice that are deficient in either IL-1α or IL-1β. (C) Incidence of disease over time. Combined data from two independent experiments. (D and E) PopLNs were harvested from 12- to 16-wk-old WT, Pstpip2^cmo, and Pstpip2^cmoxIl1β^−/− mice. Representative images of the PopLNs (D) and numbers (mean ± SEM) of popLN cells (E). Data are representative of three independent experiments with at least six mice per group. (F) Hind paw metacarpal bone H&E sections from WT, Pstpip2^cmo, and Pstpip2^cmoxIl1β^−/− mice. *P < 0.05, **P < 0.01.

Fig. 4.

IL-1β triggers downstream proinflammatory cytokine production. Levels of cytokines in the hind paws of WT, Pstpip2^cmo, and Pstpip2^cmoxIl1β^−/− mice. Each point represents an individual mouse, and the line represents the mean ± SEM. Data are representative of three independent experiments with at least eight mice per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 5.

Inflammasome-independent processing of IL-1β drives osteomyelitis. (A) Incidence of disease over time in bone marrow chimeras (donor>>recipient). (B) Secretion of IL-1β by WT or Pstpip2^cmo bone marrow-derived macrophages following stimulation with LPS, LPS+ATP, or Salmonella (Salm). (C) Incidence of disease over time for WT, Pstpip2^cmo, Pstpip2^cmoxCasp1^−/−, and Pstpip2^cmoxNlrp3^−/− mice. (D) Levels of IL-1β in the hind paws of diseased Pstpip2^cmo, Pstpip2^cmoxCasp1^−/−, and WT control mice. Each point represents an individual mouse, and the line represents the mean ± SEM. Data are combined from two independent experiments. (E) Representative micro-CT scans for WT, Pstpip2^cmo, Pstpip2^cmoxCasp1^−/−, and Pstpip2^cmoxNlrp3^−/− mice. n.s., not statistically significant. (F) Western blot analysis of IL-1β processing in the hind paws of WT, Pstpip2^cmo, Pstpip2^cmoxCasp1^−/−, and Pstpip2^cmoxIl1β^−/− mice that received footpad injections of PBS or 0.125 mg LPS 2 h before processing. Data are representative of three independent experiments. As a positive control (P.C.) for inflammasome-mediated IL-1β cleavage, WT bone marrow-derived macrophages were treated with LPS+ATP. ***P < 0.001.