Abstract

   Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease
   characterized by synovial inflammation and the production of
   autoantibodies. Previous studies have indicated an association between
   high-salt diets (HSD) and an increased risk of RA, yet the underlying
   mechanisms remain unclear. Macrophage pyroptosis, a pro-inflammatory
   form of cell death, plays a pivotal role in RA. In this study, we
   demonstrate that HSD exacerbates the severity of arthritis in
   collagen-induced arthritis (CIA) mice, correlating with macrophage
   infiltration and inflammatory lesions. Given the significant
   alterations observed in macrophages from CIA mice subjected to HSD, we
   specifically investigate the impact of HSD on macrophage responses in
   the inflammatory milieu of RA. In our in vitro experiments,
   pretreatment with NaCl enhances LPS-induced pyroptosis in RAW.264.7 and
   THP-1 cells through the p38 MAPK/NF-κB signaling pathway. Subsequent
   experiments reveal that Slc6a12 inhibitors and SGK1 silencing inhibit
   sodium-induced activation of macrophage pyroptosis and the p38
   MAPK/NF-κB signaling pathway, whereas overexpression of the SGK1 gene
   counteracts the effect of sodium on macrophages.

   In conclusion, our findings verified that high salt intake promotes the
   progression of RA and provided a detailed elucidation of the activation
   of macrophage pyroptosis induced by sodium transportation through the
   Slc6a12 channel.

   Keywords: High-salt diet, rheumatoid arthritis, macrophages,
   pyroptosis, SGK1, Slc6a12

Introduction

   Rheumatoid arthritis (RA) is a chronic systemic disorder defined by
   synovial inflammation and proliferation, concurrent with the generation
   of autoantibodies, culminating in the deterioration of cartilage and
   bone, and consequent disability [69]^1. It is hypothesized that the
   onset and progression of RA result from a multifaceted interplay of
   genetic, environmental, and life style determinants [70]^2^,[71]^3.
   This condition preferentially afflicts the geriatric female
   demographic, accounting for approximately 1% of the global population.
   Manifesting primarily through inflammation in small and medium-sized
   joints, RA substantially erodes the quality of patients' life
   [72]^4^,[73]^5. Further to this, sustained inflammation poses risks to
   additional organs, including the cardiovascular system and kidneys.
   Despite this, extant therapeutic strategies largely center on
   ameliorating inflammatory symptoms rather than providing comprehensive
   RA mitigation, and are frequently associated with a spectrum of adverse
   effects [74]^6.

   Contemporary understanding posits that the pathogenesis of bone and
   cartilage erosion in RA is predominantly due to the interactions
   involving both the innate and adaptive immune cohorts. Noteworthy is
   the aberrant activation of macrophages, innate immune cells, during the
   incipient phases of the disease [75]^7. RA-afflicted synovia witness a
   pronounced influx of these macrophages, believed to be sourced from
   both circulating monocytes and sessile tissue-resident populations.
   Monocyte-derived macrophages are progeny of hematopoietic stem cells
   located in the bone marrow, whereas resident macrophages trace their
   lineage back to embryonic development stages in the fetal liver and
   yolk sac. The phenotypic plasticity of macrophages is considerable,
   allowing them to adapt their functional stance in response to the
   evolving extracellular milieu [76]^8. Upon environmental cueing, they
   shift towards a pro-inflammatory stance, unleashing a cascade of
   inflammatory cytokines, including IL-1β, IL-12, IL-6, TNF-α and
   chemokines, which collectively conscript additional immune effector T
   and B cells to the site, thus exacerbating the inflammatory milieu
   within the joint space [77]^9.

   Pyroptosis, a lytic and inflammatory form of programmed cell death,
   primarily documented in relation to infectious disease pathologies, has
   recently garnered attention for its role in autoimmune diseases,
   including RA [78]^10^,[79]^11. Investigations have underscored the
   occurrence of macrophage pyroptosis in RA, implicating it as a driver
   in the disease's pathogenesis. This cell death pathway is distinguished
   by a swift breach of the cell membrane accompanied by the discharge of
   inflammatory mediators, most notably IL-1β and IL-18 [80]^12. The
   process is initiated when macrophages recognize exogenous danger cues
   such as lipopolysaccharide (LPS). This detection precipitates the
   assembly of the NLRP3 inflammasome, a multiprotein complex involving
   the NOD-like receptor thermal protein domain associated protein
   (NLRP3), the apoptosis-associated speck-like protein containing a
   caspase recruitment domain (ASC), and procaspase-1. Subsequent
   activation triggers the release of IL-1β and IL-18 following their
   cleavage by caspase-1. Beyond eliciting an inflammatory response,
   macrophage pyroptosis fosters the aberrant clonal expansion of
   fibroblast-like synoviocytes and the secretion of matrix
   metalloproteinases, thus magnifying tissue damage and promoting
   irreversible bone erosion in RA [81]^13^,[82]^14.

   Salt is a common seasoning that enhances the flavor of food. The World
   Health Organization recommends that sodium chloride intake should be
   less than 5 grams per person per day [83]^15. However, statistics
   indicate that the per capita salt intake in most countries is between
   9-12 grams or even higher, far exceeding the recommended amount.
   Excessive salt intake not only increases the risk of hypertension and
   cardiovascular disease, but recent studies have also demonstrated that
   a high-salt diet can lead to abnormal changes in the immune system,
   thereby increasing the risk of multiple sclerosis and systemic lupus
   erythematosus [84]^16.

   The impact of high-salt diet on the progression of RA is still
   inconclusive. A clinical study from Spain indicated that individuals
   with high sodium intake had a 50% increased risk of developing RA
   [85]^17. In this study, we have engineered a murine model to
   investigate the influence of dietary salt on collagen-induced arthritis
   (CIA) by employing DBA/1 mice. The model consisted of administering a
   high-salt diet to evaluate its impact on joint inflammation. Our
   observations reveal that mice fed with such a diet exhibited
   exacerbated joint destruction in the context of CIA. Further molecular
   investigations disclosed an upregulation of serum and
   glucocorticoid-regulated kinase 1 (SGK1) activity in response to
   elevated NaCl levels. This upregulation appeared to precipitate the
   activation of the mitogen-activated protein kinase (MAPK) p38 signaling
   pathway, which, in turn, potentiated the pyroptotic death of
   macrophages, thereby intensifying joint inflammation. These findings
   underscore the potential peril associated with high salt consumption as
   an aggravating factor for RA. Consequently, we propose adopting a
   low-salt diet as a strategic measure for individuals contending with
   RA. Additionally, our results nominate SGK1 as a promising molecular
   target for the development of innovative RA therapeutic strategies.

Materials and Methods

Clinical data

   The clinical data were sourced from patients admitted to the
   rheumatology department of the Second People's Hospital of Hefei
   between January 2022 and April 2023. The majority of patients included
   were aged between 35 and 85 years old (n=100), with a diagnosis of
   either RA or osteoarthritis (OA). Patients with a history of
   hypertension or heart disease, and those who required treatment with
   drugs containing sodium or potassium, were excluded from the study.
   Patients with RA were diagnosed using the 2010 classification criteria
   established by the American College of Rheumatology and the Federation
   of European Societies of Rheumatology. The diagnostic criteria for OA
   were in accordance with the Bone and Joint Diagnosis and Treatment
   Guidelines updated by the Orthopaedic Branch of the Chinese Medical
   Association in 2018.

Specimens

   In this study, we utilized human knee synovium tissues from RA (n = 5)
   and OA (n = 5) patients undergoing total joint replacement in the
   Department of Orthopaedics of Hefei Second People's Hospital. All
   patients provided written informed consent prior to the experiment, and
   the study protocol was approved by the Biomedical Ethics Committee of
   Anhui Medical University (Approval No. 83230267).

Animals

   Seven to eight-week-old male DBA/1 mice (average weight 18±2g) were
   procured from Gem Pharmatec (Nanjing, China) and were maintained in a
   Specific Pathogen Free (SPF) animal house, under conditions of 25°C and
   a 12-hour light-dark cycle. In the experiment, cage bias was taken into
   consideration. Instead of being fed in a separate ventilated cage, they
   were all kept in the same open environment. This study was approved by
   the Experimental Animal Ethics Committee of Anhui Medical University
   (Approval No. 20220454).

HSD treatment

   The mice were randomly assigned to either the NSD (Normal mice fed
   standard diet and provided regular drinking water) or HSD (High-salt
   diet containing 4% added NaCl and drinking water with 1% NaCl) groups
   [86]^18, with 20 mice in each group. Prior to the experiment, the mice
   in the HSD group were acclimated to the high-salt diet for one week.
   After seven days, ten mice from each group were randomly selected to
   establish the collagen-induced arthritis (CIA) model. The mice were
   then divided into four experimental groups, with ten mice per group:
   NSD + normal group (NSD), HSD + normal group (HSD), NSD + CIA model
   group (NSD + CIA), and HSD + CIA model group (HSD+CIA). Throughout the
   duration of the experiment, the body weight, dietary intake, and water
   consumption of each group were measured at two-day intervals.

CIA mouse model

   Fully emulsified chicken type II collagen (Chondrex, cat#20011, USA)
   was combined with complete Freund's adjuvant (Chondrex, cat#7023, USA)
   containing 5 mg/mL inactivated Mycobacterium tuberculosis. On day 0,
   8-week-old male DBA/1 mice received a subcutaneous injection of 100 μL
   of the emulsified collagen at the tail root. On day 21, a second
   injection of 100 μL was administered to enhance immunity. The clinical
   severity of arthritis was evaluated between 29 and 49 days after the
   initial immunization.

Evaluation of arthritis

   To assess the severity of CIA, two independent observers, unaware of
   the experimental protocol, conducted a comprehensive evaluation.
   Beginning on the 29th day after the initial immunization, the mice's
   clinical scores were measured three times every two days, including
   arthritis index, paw swelling number, and body weight. The criteria for
   arthritis index scoring were as follows: 0 = normal; 1 = erythema and
   slight swelling of the ankle joint; 2 = erythema and slight swelling
   from ankle to metatarsal or metacarpal joints; 3 = erythema and
   moderate swelling of ankle to metatarsophalangeal or metacarpal joints;
   4 = erythema and severe swelling of ankle to toe joints. The paw
   swelling number of mice in each group was also recorded every 2 days
   from day 0, with each mouse's paw consisting of 1 ankle joint and 5
   knuckles. A single spot of redness was scored as 1 point, with each
   mouse having a maximum score of 24.

Histological analysis of joint and spleen

   To ascertain the histopathological hallmarks, we employed Hematoxylin
   and Eosin (H&E) staining. Hind limbs and spleens excised from the
   subjects were initially preserved in 4% paraformaldehyde for a duration
   of 24 hours. After the fixation process, hind limbs underwent a
   decalcification protocol for 30 days, with the decalcifying solution
   replenished biweekly. In contrast, spleens were promptly processed for
   paraffin embedding. We assessed inflammatory manifestations and the
   extent of bone erosion within the knee and ankle joints by adopting a
   systematic approach that incorporated an analysis of synovial
   hypertrophy, infiltration of inflammatory cells, pannus formation, and
   the integrity of bone and cartilage. This assessment was performed
   independently by a duo of experienced investigators to ensure
   objectivity. In addition to the joint evaluation, a separate pair of
   independent researchers conducted H&E staining on spleen tissue
   samples, whereby they quantified alterations based on parameters which
   included changes in spleen morphology, the prevalence of germinal
   centers, and the degree of leukocytic infiltration.

Safranin O/Fast green staining

   Following the removal of wax, tissue sections were immersed in safranin
   O for three minutes, then briefly rinsed in distilled water for one
   minute. Fast green staining ensued for two minutes, succeeded by
   another distilled water rinse. Differentiation was achieved with a
   one-minute treatment in 1% acetic acid. Progressive dehydration was
   conducted using 95% ethanol, followed by clearance in xylene. The
   morphological alterations of the samples were then examined
   post-mounting with neutral balsam.

Mouse primary peritoneal macrophages isolation and culture

   Following euthanasia by cervical dislocation, the corpses of mice were
   sanitized through a five-minute submersion in 75% ethanol. The
   abdominal integument was retracted to reveal the peritoneal cavity. A
   cooled phosphate-buffered saline (PBS) solution was injected into the
   splenic cavity, succeeded by gentle abdominal massaging for five
   minutes to dislodge immune cells. Peritoneal exudate was then aspirated
   using a syringe armed with a 21-gauge needle. The resultant fluid was
   centrifuged at 4°C at 400×g for ten minutes to precipitate macrophages.
   These cells were then re-suspended in culture medium and enumerated.
   The macrophages were seeded in Roswell Park Memorial Institute (RPMI)
   1640 medium, procured from VivaCell in Shanghai, enhanced with 10%
   fetal bovine serum from WISENT, based in Nanjing, and buffered with an
   antibiotic-antimycotic cocktail comprising 1% penicillin and 1%
   streptomycin, both sourced from Beyotime in Shanghai. Following
   adherence to culture vessels, the cells were appropriated for
   experimental evaluations.

Cell culture and treatment

   RAW264.7 cell line (Procell Life Science & Technology Co., Ltd, Wuhan,
   China) in 10% fetal bovine serum (WISENT, China was cultured in medium
   (High sugar DMEM) (VivaCell, Shanghai, China) and placed in an
   incubator at 37°C and 5% CO[2]. Mononuclear leukemia cell line (THP-1
   cells) (Procell Life Science & Technology Co., Ltd, Wuhan, China) in
   15% fetal bovine serum (WISENT, Nanjing, China) was cultured in medium
   (RPMI 1640) (VivaCell, Shanghai, China) and placed in an incubator at
   37°C and 5% CO[2]. Prior to the experiment, THP-1 monocytes were
   transformed into M0 adherents after being treated with 100 nM
   phorbol12-myristate 13-acetate (PMA) (GLPBIO, CA, USA) on a 6-well
   plate for 48h. LPS (1μg/mL) (Solarbio, Beijing, China) in combination
   with ATP(5μM) (Sigma, USA) induces pyroptosis in Raw264.7 cells,
   whereas in THP-1-derived macrophages, pyroptosis is induced using only
   LPS. Slc6a12 inhibitor (BPDBA) (MCE, Nj, USA) was added to the six-well
   plate with a final concentration of 20μM, and the cells were stimulated
   for 24h. NaCl pretreatment was 50 mM for 12h.

Western blotting (WB)

   The total protein was separated by 10% fast polyacrylamide (PAGE) gel
   (YEASEN, Shanghai, China), and the isolated protein was transferred to
   PVDF membrane (Millipore, MA USA). Sealed with 5% skim milk at room
   temperature for 2h, then with specific primary antibody at 4°C. Rabbit
   anti-β-actin (Cat#: AF7018, Affinity) and rabbit anti-phospho-SGK1
   (Ser422) (Cat#: AF3001, Affinity) were purchased from Affinity
   Biosciences (Cincinnati, OH, USA). Rabbit anti-phospho-p38 MAPK
   (Thr180/Tyr182) (Cat#: 4511) and rabbit anti-p38 MAPK (Cat#: 8690)
   antibodies were purchased from Cell signaling technology (MA, USA).
   Anti-NF-κB p65 recombinant rabbit monoclonal antibody (Cat#:
   ET1603-12), anti-phospho-NF-κB p65(S529) recombinant rabbit monoclonal
   antibody (Cat#: ET1604-27) and anti-Gasdermin D (N terminal)
   recombinant rabbit monoclonal antibody (Cat#: HA721144) were purchased
   from HUABIO Co., LTD (Hangzhou, China). Rabbit anti-IL-18 (Cat#:
   [87]R24693) and rabbit anti-cleaved-Caspase-1 (Cat#: 341030) antibodies
   were purchased from ZEN-BIOSCIENCE (Chengdu, China). Rabbit
   anti-IL-1beta (Cat#: 66737-1-Ig), rabbit anti-SGK1 (Cat#: 28454-1-AP)
   and mouse anti-Slc6a12 (Cat#: 67700-1-Ig) antibodies were purchased
   from Proteintech group (Wuhan, China). Rabbit anti-NLRP3 (Cat#:
   WL02635) antibody was purchased by WANLEIBIO (Shenyang, China). After
   washing with PBS for 3 times, it was incubated with secondary antibody
   (Elabscience, Wuhan, China) at room temperature for 2h. After washing
   with TPBS, bands were observed with enhanced chemiluminescence reagent
   (Affinity, USA) by fully automated chemiluminescence imaging system
   (Tanon 5200, China).

Immunohistochemical staining

   Mice was sacrificed on Day49, the knee joints were fixed and
   decalcified, dehydrated with fractional alcohol, embedded in paraffin,
   and sectioned for 5µm. The tissue sections were placed in an oven at
   55°C overnight and immersed in xylene dewaxing and gradient ethanol.
   Washed with PBS 3 times for 15 minutes, washed with PBS 3 times, the
   slices were boiled in a large antigen repair solution at 100°C and
   cooled to room temperature. Wash with PBS 3 times, seal with endogenous
   peroxidase for 10 minutes, rinse with PBS 3 times, seal and serum for
   15 minutes. The primary antibody F4/80 (Cell signaling technology, MA,
   USA) was then incubated overnight at 4°C. The next day, biotin-labeled
   secondary antibody was incubated at room temperature for 20 minutes,
   washed with PBS for 3 times, labeled with horseradish peroxidase
   marker, washed with PBS for 3 times, colored with DAB, stained with
   hematoxylin, dehydrated with gradient alcohol, and mounted with neutral
   adhesive. Finally, the tissue staining results were observed in the
   digital slice scanning system Pannoramic MIDI (Pannoramic MIDI II,
   Hungary, China).

Immunofluorescence staining

   The RAW264.7 cells were adhered to a 24-well plate with a 1.5 mm
   polylysine coating. After pretreatment with NaCl (50 mM) for 12 hours,
   the RAW264.7 cells were stimulated with LPS and ATP. After 24 hours,
   the cells were fixed with acetone for 15 minutes at room temperature,
   followed by infiltration with PBS + 0.1% Triton X-100 for 15 minutes.
   Subsequently, the cells were washed three times with PBS and blocked
   with 5% BSA at room temperature for 1 hour to prevent non-specific
   antibody binding. Primary antibody staining was carried out overnight
   using NLRP3 antibody (HUABIO, Hangzhou, China) dissolved in 5% BSA at a
   1:200 ratio. A secondary antibody conjugated with Alexa Fluor 488
   (715-545-151, Jackson Immunol Research) was applied according to the
   manufacturer's instructions. The secondary antibody was incubated with
   the cells for 1 hour, followed by three washes with PBS. The cells were
   then incubated with DAPI for 10 minutes, washed three times to remove
   water stains, and finally, an anti-fluorescence quencher was applied to
   seal the slide cover. Images were obtained using a laser scanning
   confocal microscope (Leica SP8, Germany) and saved for analysis.

Scanning electron microscopy

   The RAW264.7 cells were seeded onto a 1.5 mm polylysine coated surface
   of a 24-well plate. Consistent with previous studies [88]^19, Prior to
   inducing pyroptosis, RAW264.7 cells were pretreated with NaCl (50 mM)
   for 12 hours. After 24 hours, the cells were fixed in 2% glutaraldehyde
   solution (prepared in PBS) for 2 hours. Subsequently, the samples were
   dehydrated using a series of ethanol gradients ranging from 20% to 100%
   (20%, 40%, 60%, 80%, and 100%). Each ethanol concentration was
   maintained for approximately 30 minutes. Following dehydration, the
   samples were subjected to vacuum-assisted drying with 100% acetone,
   followed by critical point drying. Finally, the samples were
   gold-plated and observed under a GeminiSEM300 (Zeiss, Germany) scanning
   electron microscope.

Flow cytometry

   Mouse paws were sectioned into 3-4 mm tissue pieces and placed in 1.5mL
   EP tubes. Hank's equilibrium salt solution (1mL), type II collagenase
   (1g/mL, 2μL), and CaCl2 solution (3mM, 5μL) were added. The mixture was
   then incubated at 37°C for 4 hours. After centrifugation and three
   washes with PBS, the cells were obtained through nylon sieving to yield
   a dispersed cell suspension. The treated single-cell suspension
   (1×10^6/100 μL) was incubated in the dark at 4°C for 30 minutes with a
   fluorescent antibody. Subsequently, the cells were centrifuged at 300
   g, washed three times with PBS, and resuspended in 300 μL of PBS for
   cell sorting observation. The isolated cells were stained with the
   following antibodies: CD45-FITC (553080, BD Pharmingen), CD19-PE
   (553786, BD Pharmingen), CD3-PE (553063, BD Pharmingen), CD4-FITC
   (553650, BD Pharmingen), CD11b-FITC (101206, biolgend), Ly6G-PE
   (551461, BD Pharmingen), and Ly6C-APC (553129, BD Pharmingen).

ELISA for cytokine measurements

   RAW264.7 macrophages were seeded in 6-well plates at a density of
   5×10^5 cells/mL. Prior to inducing pyroptosis, the cells were
   pre-incubated with NaCl (50 mM) for 12 hours. Subsequently, the cell
   culture supernatant was centrifuged at 4°C and 1500 rpm for 15 minutes
   to discard any particles. The levels of secreted IL-1β and IL-18 were
   quantified using a commercially available mouse IL-1β and IL-18 ELISA
   kit (MLBIO, Shanghai, China), following the manufacturer's
   instructions. For measurement, mouse serum samples were appropriately
   diluted.

Quantitative real-time PCR (RT-qPCR)

   Total RNA was extracted from mouse paw tissues and cells using TRIzol.
   A 20 μL total system of 5× reverse transcriptase was used for cDNA
   synthesis. The cDNA was subsequently diluted four times, and PCR
   amplification was conducted according to the manufacturer's
   instructions. The PCR reaction volume was set to 20 μL, and the
   amplification conditions were as follows: initial denaturation at 95°C
   for 5 minutes, followed by 40 cycles of denaturation at 95°C for 10
   seconds, annealing at 60°C for 35 seconds, and extension at 72°C for 15
   seconds. A melting curve analysis was performed by heating the samples
   to 95°C for 15 seconds, cooling to 60°C for 60 seconds, and then
   heating again to 95°C for 15 seconds. The expression of target genes
   was quantitatively analyzed using the 2-^ΔΔCT method, with β-actin or
   GAPDH genes used as internal references. The primer sequences for