Gasdermin D deficiency attenuates arthritis induced by traumatic injury but not autoantibody-assembled immune complexes

Abstract

Background: Gasdermin D (GSDMD) is cleaved by several proteases including by caspase-1, a component of intracellular protein complexes called inflammasomes. Caspase-1 also converts pro-interleukin-1β (pro-IL-1β) and pro-IL-18 into bioactive IL-1β and IL-18, respectively. GSDMD amino-terminal fragments form plasma membrane pores, which mediate the secretion of IL-1β and IL-18 and cause the inflammatory form of cell death pyroptosis. Here, we tested the hypothesis that GSDMD contributes to joint degeneration in the K/BxN serum transfer-induced arthritis (STIA) model in which autoantibodies against glucose-6-phosphate isomerase promote the formation of pathogenic immune complexes on the surface of myeloid cells, which highly express the inflammasomes. The unexpected outcomes with the STIA model prompted us to determine the role of GSDMD in the post-traumatic osteoarthritis (PTOA) model caused by meniscus ligamentous injury (MLI) based on the hypothesis that this pore-forming protein is activated by signals released from damaged joint tissues.

Methods: Gsdmd ^+/+ and Gsdmd^-/- mice were injected with K/BxN mouse serum or subjected to MLI to cause STIA or PTOA, respectively. Paw and ankle swelling and DXA scanning were used to assess the outcomes in the STIA model whereas histopathology and micro-computed tomography (μCT) were utilized to monitor joints in the PTOA model. Murine and human joint tissues were also examined for GSDMD, IL-1β, and IL-18 expression by qPCR, immunohistochemistry, or immunoblotting.

Results: GSDMD levels were higher in serum-inoculated paws compared to PBS-injected paws. Unexpectedly, ablation of GSDMD failed to reduce joint swelling and osteolysis, suggesting that GSDMD was dispensable for the pathogenesis of STIA. GSDMD levels were also higher in MLI compared to sham-operated joints. Importantly, ablation of GSDMD attenuated MLI-associated cartilage degradation (p = 0.0097), synovitis (p = 0.014), subchondral bone sclerosis (p = 0.0006), and subchondral bone plate thickness (p = 0.0174) based on histopathological and μCT analyses.

Conclusion: GSDMD plays a key role in the pathogenesis of PTOA, but not STIA, suggesting that its actions in experimental arthropathy are tissue context-specific.

Keywords: Arthritis; Bone; GSDMD; IL-1; Immune cells; Inflammasome; Inflammation; Pyroptosis.

Fig. 1

GSDMD deficiency did not reduce joint swelling and osteolysis induced by STIA. Six-week-old mice were injected intraperitoneally with PBS (Gsdmd^+/+: 4 females and 3 males; Gsdmd^−/−: 4 females and 2 males) or K/BxN mouse serum (Gsdmd^+/+: 3 females and 2 males; Gsdmd^−/−: 4 females and 3 males) on days 0 and 2. Ankle (A) and paw (B) thicknesses were measured daily for 12 days with a digital caliper. No differences in joint swelling were noted between male and female mice. C The hindlimbs were collected on day 12 and scanned. White arrows indicate the areas of extensive osteolysis. D, E qPCR analysis of Gsdmd mRNA expression in Gsdmd^+/+ paw tissues. Data are mean ± SD. Student’s t test; **p < 0.01; ***p < 0.001

Fig. 2

Expression of GSDMD, IL-1β, and IL-18 was increased in the articular cartilage from mice subjected to MLI and human PTOA patients. qPCR analysis of the gene expression in the articular cartilage from mice (sham surgery or MLI (A)) and human patients (control, OA, or PTOA (B, C)). The dot points reflect different patients or mouse samples. Data are mean ± SD. Student’s t test; *p < 0.05; **p < 0.01; ****p < 0.0001

Fig. 3

GSDMD was expressed in mouse joint tissues and synovium of human OA patients. A–F Representative images of specimens of Gsdmd^+/+ mice stained with GSDMD antibody. B, C, E, F Magnified views of the boxed areas in A and D. G–I Representative images of specimens of Gsdmd^−/− mice stained with GSDMD antibody. Brown and dark red show specific staining. Scale bar = 250 μm (A–I). AC, articular cartilage; BM, bone marrow; M, meniscus. J Immunoblotting analysis of GSDMD expression and cleavage in the synovium from 1 normal and 8 OA patients. GAPDH was used as a loading control

Fig. 4

GSDMD deficiency attenuated articular cartilage degeneration and synovitis. Twelve-week-old Gsdmd^+/+ and Gsdmd^−/− male mice were subjected to sham or MLI surgery. A Representative images of Safranin-O staining of the left (sham) and right (MLI) knee joints. B Representative images of anterior synovium of sham or MLI knee joints from Gsdmd^+/+ and Gsdmd^−/− mice. C OARSI and synovitis scores. OARSI scoring was performed to quantify the severity of OA. Synovitis scores are based on the severity of synovial hyperplasia and sub-synovial inflammation. Reference data for C are shown in Fig. S3A. Data are mean ± SD. N = 7–8/group. Unpaired t test; *p < 0.05; **p < 0.01. Scale bar = 250 μm

Fig. 5

GSDMD deficiency attenuated subchondral bone sclerosis. Twelve-week-old Gsdmd^+/+ and Gsdmd^−/− male mice were subjected to sham or MLI surgery. A Representative three-dimensional μCT figures of sham and MLI knee joints. “PET” color scheme from the Dragonfly software was used to highlight the subchondral bone changes. B Subchondral BV/TV changes. MLI BV/TV/Sham BV/TV percentage for each mouse was used to quantify the extent of subchondral bone sclerosis. Reference data are shown in Fig. S3B. C Subchondral bone plate thickness changes. MLI thickness/sham thickness percentage for each mouse was used to quantify the extent of subchondral bone sclerosis. Data are mean ± SD. N = 7–8/group. Unpaired t test; *p < 0.05; ***p < 0.001. Scale bar = 5 mm