13 research outputs found
Constitutive activation of NF-κB in osteoblasts, chondrocytes and stromal cells inhibits their differentiation.
<p>(A) Calvarial osteoblasts (cOB) were transduced using retroviral constructs pMX-GFP and pMX-IKK2-ca and incubated for the time points indicated. Western blots were used to show expression of Flag-tagged IKK2 and β-actin in cOB. (B) Expression of IκB and phospho-IκB in lysates of OBs transduced with pMx-GFP or the various forms of IKK2 as indicated. (C–D) Calvarial OBs (C), ST2 and ATDC5 cells (D) were infected with GFP, IKK2WT, IKK2ca, IKK2KD, or IKK2SSAA and incubated for 21 days with 10 ug/ml of bovine insulin (media was changed and supplemented with fresh insulin every 48 hrs). Cells were then stained with either alkaline phosphatase (ALP) or Alcian blue. Note reduced staining in IKK2ca conditions. Lower left panel (C) depicts optical density quantification of upper panel. Right panel (D) represents quantification of ST2 cell and ATDC5 staining (not shown) using arbitrary units expressed as % of control. Experiments were repeated at least three times in triplicate conditions. Asterisk represents p<0.01.</p
Mice expressing IKK2ca exhibit abnormal skeletal development.
<p>(A) cOB cells were harvested form newborn mice, lysed and subjected to Western blot with Flag (IKK2) and beta-actin antibodies. (B) Neurocranium of Newborn WT, Col2Cre+/IKK2ca heterozygotes (het) and homozygotes (KI). Sutures and fontanelles are widened in Col2Cre+/IKK2ca homozygotes (asterisk). Arrows (bottom) indicate the unfused and smaller supraoccipital bone in Col2Cre+/IKK2ca heterozygotes and homozygotes, respectively. Shortened snout in homozygous knock-in (large arrow) compared with hets and WT littermates. (C–D) Newborn pups were stained with Alcian blue/Alizarin red. Bone is stained red and cartilage blue. Limbs showed reduced length of scapula in newborn Col2Cre+/IKK2ca heterozygotes and homozygotes (D) Smaller skeleton and deformed vertebrate in newborn Col2Cre+/IKK2ca heterozygotes (wf) and homozygotes (ff). Reduced or missing digit ossification in Col2Cre+/IKK2ca heterozygotes and homozygotes respectively (arrows and asterisks). Reduced or diminished ossification in vertebral bodies and skull in knock-in mice is apparent (arrow heads). (E) Dorsoventral view of vertebra showing reduced width of neural arches as well as reduced or diminished degrees of ossification in vertebral bodies (arrow) of newborn Col2Cre+/IKK2ca heterozygotes and homozygotes respectively compared to WT littermates.</p
Proposed schematic model.
<p>IKK2ca mimics persistent NF-κB activation by inflammatory signals. In this model, however, activation of the NF-κB is restricted to the mesenchymal compartment using Coll2-cre driven expression of the active IKK2 form. IKK2ca leads to exacerbated expression of NF-κB subunits (indicated here as p50/p65). According to our data, NF-κB activation inhibits expression of osteogenic markers while stimulating expression of anti-osteogenic (SOST/sclerostin, DKK1) that antagonize Wnt binding to LRP and increasing pro-osteoclastogenic factors (RANKL) that stimulate osteoclast formation. The net effect of these actions is inhibition of bone formation and increased bone resorption.</p
Expression of IKK2ca impedes bone formation and reduces bone mineral density.
<p>(A) Micro-CT analysis of four-week old Col2Cre+/IKK2ca heterozygote (Het) and WT littermate (n = 6/group). Col2Cre+/IKK2ca heterozygote has reduced trabecular bone in proximal tibia and femur compared to WT littermate. Quantitation of bone volume (BV) over total volume (TV), trabecular number (Tb.N), trabecular thickness (Tb.Th.) and trabecular separation (Tb.Sp.) is shown. (B) Bone formation rate - 4-week old mice (n = 6/group) were injected with two consecutive labels (arrows) of calcein (7 days apart) to measure bone formation rate. Asterisk represents p<0.05.</p
Gasdermin D mediates the pathogenesis of neonatal-onset multisystem inflammatory disease in mice
<div><p>Mutated NLRP3 assembles a hyperactive inflammasome, which causes excessive secretion of interleukin (IL)-1β and IL-18 and, ultimately, a spectrum of autoinflammatory disorders known as cryopyrinopathies of which neonatal-onset multisystem inflammatory disease (NOMID) is the most severe phenotype. NOMID mice phenocopy several features of the human disease as they develop severe systemic inflammation driven by IL-1β and IL-18 overproduction associated with damage to multiple organs, including spleen, skin, liver, and skeleton. Secretion of IL-1β and IL-18 requires gasdermin D (GSDMD), which—upon activation by the inflammasomes—translocates to the plasma membrane where it forms pores through which these cytokines are released. However, excessive pore formation resulting from sustained activation of GSDMD compromises membrane integrity and ultimately causes a pro-inflammatory form of cell death, termed pyroptosis. In this study, we first established a strong correlation between NLRP3 inflammasome activation and GSDMD processing and pyroptosis in vitro. Next, we used NOMID mice to determine the extent to which GSDMD-driven pyroptosis influences the pathogenesis of this disorder. Remarkably, all NOMID-associated inflammatory symptoms are prevented upon ablation of GSDMD. Thus, GSDMD-dependent actions are required for the pathogenesis of NOMID in mice.</p></div
Unfractionated NOMID bone marrow cells proliferate and survive significantly less than their WT counterparts.
<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
OC differentiation is increased in NOMID cells.
<p>Unfractionated bone marrow cells (<i>A</i>) or BMM previously cultured for 3 days in the presence of M-CSF (<i>B</i>) were induced to differentiate into OC in the presence of M-CSF and 100 ng/ml RANKL. (<i>C</i>) Co-cultures of WT or NOMID (NOM) BMM and WT BMSC were carried out in the presence of 10 nM dexamethasone and 1 nM 1,25(OH)<sub>2</sub> vitamin D<sub>3</sub> for 5–7 days. The cultures were stained for TRAP activity, and the number of OC (cells stained in red with ≥3 nuclei, arrow) were counted manually. The pictures were taken at the same magnification (×4) for both genotypes. The data show that NOMID cells formed more OC than WT cells. Determinations were performed in triplicate and expressed as the mean ± S.E.M. Results are representative of at least three independent experiments. **P<0.007.</p
NOMID mice exhibit disorganized growth plates.
<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
NOMID mice exhibit stunted skeletal growth and reduced bone mass.
<p>X-ray radiography (<i>A</i>), DXA (<i>B</i>) or μCT analysis of the femur (<i>C, E</i>–<i>H</i>) of NOMID and WT mice at age P13. DXA and μCT 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.
<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