Pyroptosis by NLRP3/caspase‐1/gasdermin‐D pathway in synovial tissues of rheumatoid arthritis patients

Abstract We investigated the potential involvement of pyroptosis, a proinflammatory form of regulated cell death, in rheumatoid arthritis (RA). Synovial fluid, synovial tissues and/or serum were compared among 32 patients with RA, 46 patients with osteoarthritis (OA) and 30 healthy controls. Samples were assayed for interleukin (IL)‐1β, IL‐18 and lactate hydrogenase (LDH). Synovial expression of NLRP3, caspase‐1 and cleaved gasdermin D (GSDMD) was assayed using immunohistochemistry and multiplex immunohistochemistry. Patients with RA showed significantly higher levels of IL‐1β and IL‐18 in synovial fluid than patients with OA, and significantly higher levels of both cytokines in serum than healthy controls. RA was associated with higher levels of LDH in synovial fluid than OA. Among patients with RA, levels of IL‐1β, IL‐18 and LDH were significantly higher in synovial fluid than in serum, and the levels in synovial fluid positively correlated with disease activity and inflammation. Synovial cells, particularly macrophages, showed upregulation of NLRP3, caspase‐1 and cleaved GSDMD in RA compared to OA. Our results implicate pyroptosis in the pathogenesis of RA, perhaps as a driver of local inflammation in joints.

gasdermin D (GSDMD) was assayed using immunohistochemistry and multiplex immunohistochemistry. Patients with RA showed significantly higher levels of IL-1β and IL-18 in synovial fluid than patients with OA, and significantly higher levels of both cytokines in serum than healthy controls. RA was associated with higher levels of LDH in synovial fluid than OA. Among patients with RA, levels of IL-1β, IL-18 and LDH were significantly higher in synovial fluid than in serum, and the levels in synovial fluid positively correlated with disease activity and inflammation. Synovial cells, particularly macrophages, showed upregulation of NLRP3, caspase-1 and cleaved GSDMD in RA compared to OA. Our results implicate pyroptosis in the pathogenesis of RA, perhaps as a driver of local inflammation in joints.

K E Y W O R D S
caspase-1, gasdermin D, NLRP3, pyroptosis, rheumatoid arthritis, synovial tissue Here we explored the implication of pyroptosis in RA by comparing levels of IL-1β, IL-18 and LDH in serum or synovial fluid among patients with RA, patients with osteoarthritis (OA) and healthy controls. We also examined expression of NLRP3, caspase-1 and GSDMD-N in the synovium. Necroptosis is another type of regulated cell death, and caspase-1 does not participate in the process. Moreover, IL-1β and IL-18 are long considered signatures of pyroptotic cell death and GSDMD executes pyroptosis. 12 These indicators help to distinguish pyroptosis from necroptosis.
Since OA arises from mechanical injury and involves milder inflammation and tissue damage than RA, 13 we compared synovium from patients with OA to that from patients with RA. We hope, through the human specimens, to provide clinical evidence of the role of pyroptosis in RA.

| Samples
The study protocol was approved by the ethics committee of Luoyang Orthopaedic Hospital (KY2022-001-02). Synovial tissue and synovial fluid were sampled from 32 patients with RA and 46 patients with OA who underwent knee arthroplasty in our department of orthopaedic surgery between October 2020 and June 2022. RA was diagnosed based on the 2010 criteria of the American College of Rheumatology and European League Against Rheumatism, 14 while OA was diagnosed based on the most recent criteria of the European League Against Rheumatism. 15 In addition, we collected serum from the 32 patients with RA on the day before surgery, as well as from 30 healthy controls who visited our clinic for routine outpatient exams. We excluded subjects who received oral prednisolone ≥10 mg daily or a glucocorticoid injection within the previous 3 months. 16 All participants signed written informed consent before participating in the study.
The following clinical data were prospectively collected from patients as appropriate: clinicodemographic characteristics, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and score on the 'Disease Activity Score in 28 Joints-ESR' (DAS28-ESR) scale. 17
Samples were incubated for 90 min in 96-well plates that had already been precoated with the desired antibody. The plate was washed, incubated with biotinylated detection antibody for 60 min, washed again, incubated with enzyme conjugate for 30 min, washed again and incubated with chromogenic substrate followed by stop solution. The LDH level in synovial fluid and serum was assayed using a commercial kit (A020-2, Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's instructions. In all assays, absorbance at 450 nm was measured using a microplate reader (BioTek,).

| Histopathology
Synovial tissue was fixed with 4% paraformaldehyde, embedded in paraffin, sliced to a thickness of 4 μm, and stained with haematoxylin and eosin. Tissues were viewed under a BX40 light microscope (Olympus) and photographed using a digital camera (Olympus), and histopathology was assessed as described. 18 The average number of cells per 1 mm 2 was determined from three randomly selected fields at a magnification of 400×. The thickness of the synovial lining layer was determined by averaging the number of cells in three fields randomly selected in longitudinal tissue sections at a magnification of 400 × .

| Immunohistochemistry
Paraffin-embedded synovial tissues were also analysed using im-

| Multiplex immun ohi sto che mistry
Paraffin-embedded synovial tissue sections were subjected to multiplex immunohistochemistry. These sections were deparaffinized, rehydrated and incubated in 3% H 2 O 2 as described above, then washed in 1× Tris-buffered saline containing 0.5% Tween-20 (TBST) and subjected to multiplex staining using a commercial kit (catalogue no. NEL861001KT, Akoya Bioscience) according to the manufacturer's instructions. Sections were subjected to antigen retrieval, blocked in blocking solution at room temperature, then incubated with primary antibody against the macrophage marker CD68 (1:800 dilution; catalogue no. 76437, CST) or with the same primary antibodies described above against NLRP3, caspase-1 or GSDMD-N. Sections were rinsed in TBST, incubated with horseradish peroxidase-conjugated secondary antibody and incubated with Opal. The once-stained sections were again subjected to antigen retrieval following by staining with the subsequent antibody in the multiplex as described above. Finally, nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI), sections were washed with TBST and fluorescent anti-quencher was added.
Multispectral fluorescence images were acquired using the Vectra Polaris Automated Quantitative Pathology Imaging System (Akoya Biosciences).

| S TATIS TIC AL ANALYS IS
Data were reported as mean ± SD and analysed statistically using SPSS 22.0 (IBM, Chicago, IL, USA). Intergroup differences in continuous variables were assessed for significance using Student's t test or the Mann-Whitney test. Differences in categorical variables were assessed using a chi-squared test. Potential associations between variables were analysed using bivariate Pearson correlation. Differences associated with p < 0.05 were considered statistically significant.

| Characteristics of patients and controls
Characteristics of the study participants are summarized in

| Pyroptosis-related cytokines and LDH
Patients with RA showed significantly higher levels of IL-1β and IL-18 in synovial fluid than patients with OA (Figure 2A,B), and they showed significantly higher levels of both cytokines in serum than healthy controls did ( Figure 2D

| Associations between RA and synovial expression of NLRP3, caspase-1 and GSDMD-N
Immunohistochemistry and multiplex immunohistochemistry revealed upregulation of NLRP3, caspase-1 and GSDMD-N in the synovium of patients with RA compared to the corresponding tissue from patients with OA ( Figure 4A,B). This upregulation was observed in various types of synovial cells, especially macrophages, which we identified using the marker CD68 ( Figure 4B). Pyroptosis is known to occur mainly in macrophages and other phagocytes of the myeloid lineage, and the pathogenesis of RA is known to be driven by macrophages. 6,19 Our results are consistent with the increasement of synovial pyroptosis in RA which may create a hyper-inflammatory microenvironment around joints. promote pyroptosis. 34 We speculate it may be that some PAMPs or DAMPs may upregulate NLRP3 but not promote pyroptosis in macrophages.

F I G U R E 4
Expression of NLRP3, caspase-1 and gasdermin D-N-terminal (GSDMD-N) in synovial tissues from patients with osteoarthritis (OA) or rheumatoid arthritis (RA). (A) Sections were stained with antibodies against the indicated proteins. Scale bar, 100 μm. (B) Sections were stained with an antibody against the macrophage marker CD68 and another antibody against a pyroptosis-related marker. Nuclei were counterstained with DAPI. Scale bar, 50 μm.

F I G U R E 5
Schematic showing how pyroptosis may contribute to the pathogenesis of rheumatoid arthritis. In synovial cells, particularly macrophages, activation of the NLRP3/caspase-1/gasdermin D pathway leads to pyroptosis. Caspase-1 activates IL-1β and IL-18, which are released into the synovial fluid, while gasdermin D forms pores in the plasma membrane, leading to cytoplasmic swelling and ultimately to lysis, which releases lactate dehydrogenase and other cell contents into the synovial fluid.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data of the study was available upon reasonable request from the corresponding author.