Constitutively activated NLRP3 inflammasome causes inflammation and abnormal skeletal development in mice

Abstract

The NLRP3 inflammasome complex is responsible for maturation of the pro-inflammatory cytokine, IL-1β. Mutations in NLRP3 are responsible for the cryopyrinopathies, a spectrum of conditions including neonatal-onset multisystem inflammatory disease (NOMID). While excessive production of IL-1β 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.

Figure 1. NOMID mice exhibit growth retardation, perinatal death and inflammation in the joints.

Body weight (A) and survival (B) 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. (C) 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).

Figure 2. NOMID mice develop leukocytosis associated with high levels of inflammatory mediators.

Complete blood cell counts (A) and serum cytokine analysis (B) 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.

Figure 3. NOMID mice exhibit stunted skeletal growth and reduced bone mass.

X-ray radiography (A), DXA (B) or μCT analysis of the femur (C, E–H) 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 (C, arrowhead) in 2 NOMID mice. (D) The metaphyseal region containing (E) or contiguous (F) to the spike (depicted in black in Fig. 3D) 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 (G) and thinner cortical bone (H). 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.

Figure 4. NOMID mice exhibit disorganized growth plates.

Femoral sections from P13 (A–C) or P8 (D and E) mice were used for safranin O (A) and H&E (B, D and E) staining or for TUNEL (C). Original magnification: ×20 (A and C), ×10 (B, D and E). 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.

Figure 5. Type II collagen staining is reduced in the acellular structure in NOMID mice.

Femoral sections from P8 (A, B and C) or P13 mice (D) were stained for types I, X (green), II (red) collagen, and counterstained with DAPI (blue). Type II collagen staining was not observed in the acellular structure within the cartilage zone above the hypertrophic chondrocyte zone (hz). Original magnification: ×20 (A and B), ×10 (C and D).

Figure 6. Bone resorption is increased in NOMID mice.

Femoral sections from P13 mice were stained with H&E (A) or for TRAP activity (B–D), and OC number (C) or surface (D) per bone surface was determined. NOMID mice exhibited a larger bone marrow cavity in relation to overall bone size compared to WT mice (A). Abundant OC (stained in red, B) 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 (A), ×10 (B, trabecular region), ×20 (B, cortical region). Serum was collected for the measurement of CTX-1 levels (E), and supernatants were collected from centrifuged bone marrow for the measurement of IL-1β (F). CTX-1 and IL-1β 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. (C, D and F) or mean ± S.E.M. (E). *P<0.05; **P<0.007.

Figure 7. NOMID mice exhibit inflammation in the bone marrow, and bone cells express NLRP3.

Bone marrow cells were unstained (A and B) or stained with antibodies against CD11b, Gr1 or CD117 (C and D). The expression of CD117 was analyzed by gating cells expressing low levels (E) or high levels (F) of CD11b and Gr1. The number of CD11b^low/Gr1^low/CD117⁺ cells, but not CD11b^high/Gr1^high/CD117⁺ cells, was increased in NOMID mice. Flow cytometry experiments were repeated up to 4 times with similar results. (G) BMM were induced to differentiate into OC in the presence of M-CSF and 100 ng/ml RANKL for the indicated times, and some cultures were further stimulated with 100 ng/ml LPS for 24 hours. (H) BMSC were induced to differentiate into OB for 1 or 7 days, and some cultures were then stimulated with 20 ng/ml TNF-α for 24 hours. Western blot analysis shows that NLRP3 expression was maintained throughout cell differentiation and was up-regulated by LPS or TNF-α. β-actin was used as a protein loading control.

Figure 8. OC differentiation is increased in NOMID cells.

Unfractionated bone marrow cells (A) or BMM previously cultured for 3 days in the presence of M-CSF (B) were induced to differentiate into OC in the presence of M-CSF and 100 ng/ml RANKL. (C) 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)₂ vitamin D₃ 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.

Figure 9. Unfractionated NOMID bone marrow cells proliferate and survive significantly less than their WT counterparts.

Unfractionated bone marrow cells (A–C) or BMM (D and E) were cultured in media containing M-CSF for the indicated times. Proliferation (A and D), metabolic activity (B and E) and Western blot analysis of PARP cleavage (C) 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).