14 research outputs found

    NLRP12 provides a critical checkpoint for osteoclast differentiation

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    Members of the nucleotide-binding leucine-rich repeat-containing receptor (NLR) family are generally thought of as initiators of inflammation and are important in a number of inflammatory diseases. However, recent evidence has started to emerge that several NLRs can serve as checkpoint proteins against specific inflammatory pathways. Although checkpoint proteins are well accepted for their importance in adaptive immunity, their roles in innate immunity are still nascent. Receptor activator of nuclear factor kappa B ligand (RANKL), a tumor necrosis factor family cytokine responsible for basal and most forms of pathologic osteoclastogenesis, sends important differentiation signals through the alternative nuclear factor kappa B pathway. This report shows that an NLR member, nucleotide-binding leucine-rich repeat and pyrin domain-containing receptor 12, provides a brake on the activity of RANKL even in noninflammatory settings, extending the role for this type of NLR beyond inflammation-related disease

    Constitutively Activated NLRP3 Inflammasome Causes Inflammation and Abnormal Skeletal Development in Mice

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    The NLRP3 inflammasome complex is responsible for maturation of the pro-inflammatory cytokine, IL-1ÎČ. Mutations in NLRP3 are responsible for the cryopyrinopathies, a spectrum of conditions including neonatal-onset multisystem inflammatory disease (NOMID). While excessive production of IL-1ÎČ and systemic inflammation are common to all cryopyrinopathy disorders, skeletal abnormalities, prominently in the knees, and low bone mass are unique features of patients with NOMID. To gain insights into the mechanisms underlying skeletal abnormalities in NOMID, we generated knock-in mice globally expressing the D301N NLRP3 mutation (ortholog of D303N in human NLRP3). NOMID mice exhibit neutrophilia in blood and many tissues, including knee joints, and high levels of serum inflammatory mediators. They also exhibit growth retardation and severe postnatal osteopenia stemming at least in part from abnormally accelerated bone resorption, attended by increased osteoclastogenesis. Histologic analysis of knee joints revealed abnormal growth plates, with loss of chondrocytes and growth arrest in the central region of the epiphyses. Most strikingly, a tissue “spike" was observed in the mid-region of the growth plate in the long bones of all NOMID mice that may be the precursor to more severe deformations analogous to those observed in NOMID patients. These findings provide direct evidence linking a NOMID-associated NLRP3-activating mutation to abnormalities of postnatal skeletal growth and bone remodeling

    NLRP12 provides a critical checkpoint for osteoclast differentiation

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    The alternative or noncanonical nuclear factor kappa B (NF-ÎșB) pathway regulates the osteoclast (OC) response to receptor activator of nuclear factor kappa B ligand (RANKL) and thus bone metabolism. Although several lines of evidence support the emerging concept that nucleotide-binding leucine-rich repeat and pyrin domain-containing receptor 12 (NLRP12) impedes alternative NF-ÎșB activation in innate immune cells, a functional role for NLRP12 outside an inflammatory disease model has yet to be reported. Our study demonstrates that NLRP12 has a protective role in bone via suppression of alternative NF-ÎșB–induced osteoclastogenesis and is down-modulated in response to osteoclastogenic stimuli. Here, we show that retroviral overexpression of NLRP12 suppressed RelB nuclear translocation and OC formation. Conversely, genetic ablation of NLRP12 promoted NIK stabilization, RelB nuclear translocation, and increased osteoclastogenesis in vitro. Using radiation chimeras, we demonstrated these in vitro observations dovetail with our in vivo findings that NLRP12 deficiency leads to enhanced OC numbers accompanied by a significant decline in bone mass under physiological conditions. Consistent with the basal bone phenotype, we also observed an enhanced osteolytic response following RANKL injection over the calvaria of NLRP12-deficient chimeric mice compared with wild-type control mice. Thus, modulation of NLRP12 levels controls alternative NF-ÎșB signaling in OC precursors, altering bone homeostasis and osteolytic responses

    NOMID mice exhibit disorganized growth plates.

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    <p>Femoral sections from P13 (<i>A</i>–<i>C</i>) or P8 (<i>D</i> and <i>E</i>) mice were used for safranin O (<i>A</i>) and H&E (<i>B</i>, <i>D</i> and <i>E</i>) staining or for TUNEL (<i>C</i>). Original magnification: ×20 (<i>A</i> and <i>C</i>), ×10 (<i>B</i>, <i>D</i> and <i>E</i>). The spike (arrowhead) and early morphological changes (D, arrowhead) were observed only in NOMID mice. NOMID cells showed a high degree of apoptosis. hz, hypertrophic zone.</p

    Bone resorption is increased in NOMID mice.

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    <p>Femoral sections from P13 mice were stained with H&E (<i>A</i>) or for TRAP activity (<i>B</i>–<i>D</i>), and OC number (<i>C</i>) or surface (<i>D</i>) per bone surface was determined. NOMID mice exhibited a larger bone marrow cavity in relation to overall bone size compared to WT mice (<i>A</i>). Abundant OC (stained in red, <i>B</i>) were present in the primary spongiosa and on the endocortical bone surface of NOMID mice. There were fewer trabeculae (T) and thinner cortical bone (bracket) in NOMID compared to WT mice. Original magnification: ×2 (<i>A</i>), ×10 (<i>B</i>, trabecular region), ×20 (<i>B</i>, cortical region). Serum was collected for the measurement of CTX-1 levels (<i>E</i>), and supernatants were collected from centrifuged bone marrow for the measurement of IL-1ÎČ (F). CTX-1 and IL-1ÎČ levels were 3- to 4-fold higher in NOMID compared to WT mice. Quantitative data were obtained from 5 mice/genotype and expressed as the mean ± S.D. (<i>C</i>, <i>D</i> and <i>F</i>) or mean ± S.E.M. (<i>E</i>). *P<0.05; **P<0.007.</p

    NOMID mice exhibit stunted skeletal growth and reduced bone mass.

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    <p>X-ray radiography (<i>A</i>), DXA (<i>B</i>) or ÎŒCT analysis of the femur (<i>C, E</i>–<i>H</i>) of NOMID and WT mice at age P13. DXA and ÎŒCT 3D reconstruction revealed generalized osteopenia and the presence of a tissue spike across the growth plate (<i>C</i>, arrowhead) in 2 NOMID mice. (<i>D</i>) The metaphyseal region containing (<i>E</i>) or contiguous (<i>F</i>) to the spike (depicted in black in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035979#pone-0035979-g003" target="_blank">Fig. 3D</a>) where trabecular bone volume (BV/TV) was quantified. BV/TV was unchanged in the metaphyseal region that included the spike, but was decreased in the region contiguous to this structure. NOMID mice also exhibited significantly lower cortical area (<i>G</i>) and thinner cortical bone (<i>H</i>). Quantitative data were obtained from 5–6 mice/genotype and expressed as the mean ± S.E.M. *P<0.05; **P<0.007. BMD, bone mineral density.</p

    NOMID mice develop leukocytosis associated with high levels of inflammatory mediators.

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    <p>Complete blood cell counts (<i>A</i>) and serum cytokine analysis (<i>B</i>) were carried out on samples harvested from P12–13 mice (n = 4–7). NOMID mice developed neutrophilia, lymphopenia, thrombocytosis and anemia and produced higher levels of several inflammatory mediators. G-CSF values were extrapolated beyond the standard range. IL-6 and TNF-α levels were near statistical significance between genotypes. Data are expressed as mean ± S.E.M. *P<0.05, **P<0.007. WBC, white blood cells; RBC, red blood cells.</p

    NOMID mice exhibit growth retardation, perinatal death and inflammation in the joints.

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    <p>Body weight (<i>A</i>) and survival (<i>B</i>) were monitored daily for 3 weeks (22–25 mice/genotype). NOMID mice demonstrated significantly reduced body weight compared to WT mice by day 5, and most died by 2 weeks of age. (<i>C</i>) H&E staining of the knee joints from P8 mice. Original magnification, ×10. NOMID mice displayed massive leukocytic infiltrates in the joints and surrounding tissues (arrows).</p

    Unfractionated NOMID bone marrow cells proliferate and survive significantly less than their WT counterparts.

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    <p>Unfractionated bone marrow cells (<i>A</i>–<i>C</i>) or BMM (<i>D</i> and <i>E</i>) were cultured in media containing M-CSF for the indicated times. Proliferation (<i>A</i> and <i>D</i>), metabolic activity (<i>B</i> and <i>E</i>) and Western blot analysis of PARP cleavage (<i>C</i>) were carried out. While unfractionated NOMID cells proliferated and survived less than WT cells, no differences were seen in BMM proliferation and survival between genotypes. PARP cleavage was higher in NOMID than in WT cells. Determinations were performed in triplicate and expressed as the mean ± S.D. Results are representative of three independent experiments. *P<0.05; **P<0.007 over the control (Ctl).</p
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