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Abstract
Objectives Methylprednisolone (mPSL) pulse therapy is an essential option for patients with active systemic lupus erythematosus, but there is a risk of adverse events related to microcirculation disorders, including idiopathic osteonecrosis of the femoral head (ONFH). Recent studies have revealed that excessive neutrophil extracellular traps (NETs) are involved in microcirculation disorders. This study aimed to demonstrate that mPSL pulse could induce NETs in lupus mice and identify the factors contributing to this induction.
Methods Six mice with imiquimod (IMQ)-induced lupus-like disease and six normal mice were intraperitoneally injected with mPSL on days 39 to 41, and five mice with IMQ-induced lupus-like disease and six normal mice were injected with phosphate-buffered saline. Pathological examinations were conducted to evaluate the ischaemic state of the femoral head and tissue infiltration of NET-forming neutrophils. Proteome analysis was performed to extract plasma proteins specifically elevated in mPSL-administered mice with IMQ-induced lupus-like disease, and their effects on NET formation were assessed in vitro.
Results Mice with IMQ-induced lupus-like disease that received mPSL pulse demonstrated ischaemia of the femoral head cartilage with tissue infiltration of NET-forming neutrophils. Proteome analysis suggested that prenylcysteine oxidase 1 (PCYOX1) played a role in this phenomenon. The reaction of PCYOX1-containing very low-density lipoproteins (VLDL) with its substrate farnesylcysteine (FC) induced NETs in vitro. The combined addition of IMQ and mPSL synergistically enhanced VLDL-plus-FC-induced NET formation.
Conclusion PCYOX1 and related factors are worthy of attention to understand the underlying mechanisms and create novel therapeutic strategies for mPSL-mediated microcirculation disorders, including ONFH.
- Lupus Erythematosus, Systemic
- Glucocorticoids
- Lipids
Data availability statement
Data are available on reasonable request.
This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.
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WHAT IS ALREADY KNOWN ON THIS TOPIC
Methylprednisolone (mPSL) pulse therapy is an essential option for patients with active systemic lupus erythematosus (SLE), but there is a risk of adverse events related to microcirculation disorders, including idiopathic osteonecrosis of the femoral head (ONFH).
Excessive neutrophil extracellular traps (NETs) are involved in microcirculation disorders.
Prenylcysteine oxidase 1 (PCYOX1), which is found in abundance in very low-density lipoproteins (VLDL), reacts with farnesylcysteine (FC) as a substrate to produce hydrogen peroxide (H2O2) and farnesal.
WHAT THIS STUDY ADDS
Mice with IMQ-induced lupus-like disease that received mPSL pulse demonstrated ischaemia of the femoral head cartilage with tissue infiltration of NET-forming neutrophils.
The reaction of PCYOX1-containing VLDL with its substrate FC induced NETs in vitro.
The combined addition of IMQ and mPSL synergistically enhanced VLDL-plus-FC-induced NET formation.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
PCYOX1 and related factors are worthy of attention to understand the underlying mechanisms and create novel therapeutic strategies for mPSL-mediated microcirculation disorders, including ONFH.
Introduction
Systemic lupus erythematosus (SLE) is an autoimmune disease characterised by tissue deposition of immune complexes, resulting in various disorders of systemic organs.1 Treatment consists of oral administration of glucocorticoids at 0.5 mg/kg/day to 1 mg/kg/day. Besides, methylprednisolone (mPSL) at 0.5 g/day to 1 g/day is given intravenously for 3 days to patients with active SLE. This mPSL application is called mPSL pulse therapy. Although the therapeutic efficacy of mPSL pulse therapy is undoubted, there is a risk of adverse events related to microcirculation disorders, including idiopathic osteonecrosis of the femoral head (ONFH).2
In ONFH, blood flow to the femoral head is non-traumatically interrupted, resulting in osteocyte necrosis.3 A recent study has suggested that neutrophil extracellular traps (NETs) are involved in ONFH development.4 NETs are web-like extracellular substances composed of DNA and neutrophil cytoplasmic granules released from activated neutrophils.5 Although NETs are essential for innate immunity, NET accumulation in tissues due to their excessive formation or retarded degradation can cause cell injury, autoimmune diseases and microcirculation disorders owing to their thrombogenicity.6
This study investigated the effects of mPSL pulse on ONFH development in a lupus-like murine model. Mice with imiquimod (IMQ)-induced lupus-like disease7 were used in this study. IMQ is a Toll-like receptor 7 agonist and can induce type I interferon, resulting in the production of autoantibodies, including anti-double-stranded DNA (anti-dsDNA) antibody, and IC-mediated lupus-like disorders. After receiving mPSL pulse, mice with IMQ-induced lupus-like disease demonstrated ischaemia of the femoral head cartilage with infiltration of NET-forming neutrophils in small vessels surrounding the femoral head, although osteocyte necrosis was not evident. NET formation increased in mice with IMQ-induced lupus-like disease compared with controls and enhanced by mPSL pulse.
Proteome analysis of plasma, followed by a literature review, suggested that prenylcysteine oxidase 1 (PCYOX1) could induce NET formation in mPSL-administered mice with IMQ-induced lupus-like disease. PCYOX1 reacts with farnesylcysteine (FC) as a substrate to produce hydrogen peroxide (H2O2) and farnesal.8 9 It is found in abundance in lipoproteins, especially in very low-density lipoproteins (VLDL).9 10 This study demonstrated that the reaction of VLDL with FC induces NETs and that the combined addition of IMQ and mPSL enhances VLDL-plus-FC-mediated NET formation in vitro. mPSL pulse for lupus can induce NET formation, possibly resulting in microcirculation disorders.
Materials and methods
Mice with lupus-like disease
Mice with IMQ-induced lupus-like disease were used. In accordance with a previous method,7 5% IMQ cream (Mochida Pharmaceutical, Tokyo, Japan) was rubbed into one auricle of 8-week-old female BALB/c mice (n=11) three weekly for 8 weeks (1.25 mg IMQ/site). BALB/c mice without IMQ treatment (8-week-old females; n=12) were employed as controls. Blood was collected by tail cut under anaesthesia on days 28 and 56.
Measurement of anti-dsDNA antibody titre
Plasma was collected by centrifugation of day 28 blood, and the anti-dsDNA antibody titre was determined using an ELISA kit (Fujifilm Wako Shibayagi, Gunma, Japan).
mPSL pulse
After anti-dsDNA antibody production had been confirmed in mice that received IMQ, they were allocated to two groups. Six mice were intraperitoneally injected with mPSL (Pfizer Japan, Tokyo, Japan) on days 39 to 41 (20 mg/kg/1 mL phosphate-buffered saline (PBS)), whereas the other five mice were intraperitoneally injected with 1 mL PBS. BALB/c mice without IMQ treatment were also allocated to two groups and treated similarly (n=6 each). These treatments classified mice into group A: IMQ-unapplied mice without mPSL pulse (n=6), group B: IMQ-applied mice without mPSL pulse (n=5), group C: IMQ-unapplied mice with mPSL pulse (n=6) and group D: IMQ-applied mice with mPSL pulse (n=6).
Pathological analysis
All mice were euthanised by exsanguination under anaesthesia for pathological analysis on day 56. Systemic organs were formalin-fixed, paraffin-embedded and sliced into 4 µm sections for H&E and periodic acid Schiff (PAS) staining. One kidney was frozen and sliced into 4 µm sections for immunofluorescence staining for IgG and NETs. For IgG staining, sections fixed with cold acetone for 5 min were soaked in Protein Block Serum-Free (Dako, Glostrup, Hovedstaden, Denmark) for 10 min to prevent non-specific antibody binding. Thereafter, the sections were reacted with Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (1:1000 dilution; Abcam, Cambridge, UK) in the dark for 60 min at room temperature (RT). Alternatively, sections fixed with 4% paraformaldehyde (PFA; BioLegend, San Diego, California) for 15 min at RT were reacted first with goat anti-myeloperoxidase (MPO) antibody (1:100 dilution; R&D Systems, Minneapolis, Minnesota) overnight at 4℃ and for 60 min at RT with rabbit anti-citrullinated histone H3 (Cit-H3) antibody (1:50 dilution; Abcam) as a NET marker. The sections were reacted with Alexa Fluor 488-conjugated donkey anti-goat IgG antibody (1:500 dilution; Abcam) and Alexa Fluor 594-conjugated donkey anti-rabbit IgG antibody (1:500 dilution; Abcam) for 60 min in the dark at RT. Finally, the sections were mounted with VECTASHIELD Mounting Medium with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, California) and observed under a fluorescence microscope.
Immunohistochemistry for hypoxia-inducible factor 1α
Formalin-fixed and paraffin-embedded (FFPE) tissue sections were autoclaved at 121℃ for 10 min with citrate buffer (pH 6.0) to retrieve antigens and exposed to methanol supplemented with 3% H2O2 for 10 min to consume endogenous peroxidase in tissues. After nonspecific binding of antibodies blocked by 10% goat serum, the sections were reacted with mouse anti-hypoxia-inducible factor 1α (HIF-1α) antibody (1:100 dilution; R&D Systems) for 60 min at RT. After removal of unbound antibodies with PBS, the sections were reacted with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (1:50 dilution; Seracare, Milford, MA, USA) for 60 min at RT. HRP activity was developed with diaminobenzidine. The Allred scoring system11 was adopted for the semiquantitative evaluation of HIF-1α expression. In brief, the proportion score (PS) and intensity score (IS) were added to obtain the total score. The PS was defined according to the proportion of positive cells: 0 as none, 1 as 0 to 1/100, 2 as 1/100 to 1/10, 3 as 1/10 to 1/3, 4 as 1/3 to 2/3, and 5 as 2/3 to 1. The IS was defined according to the staining intensity: 0 as negative, 1 as weak, 2 as intermediate, and three as strong.
Proteome analysis
Plasma collected on day 56 was pooled in each group and subjected to shotgun analysis. The analysis was entrusted to the Chemical Evaluation and Research Institute (Tokyo, Japan).
H2O2 production by reaction of PCYOX1 with FC
Recombinant human PCYOX1 (60 µg/mL; Abcam) was incubated with its substrate FC (200 µM; Avanti Polar Lipids, Alabaster, AL, USA) at 37°C for 4 hour. In other experiments, VLDL (0, 6.25, 12.5, 25, and 50 µg; 88–95% lipid and 5%–12% protein; Sigma-Aldrich, St. Louis, MO, USA) was used instead of PCYOX1. H2O2 production was measured using a kit (ENZO Life Sciences, Farmingdale, NY, USA).
NET induction by H2O2 and farnesal
Neutrophils isolated from peripheral blood of healthy volunteers using Polymorphprep (Serumwerk Bernburg AG, Bernburg, Germany) were suspended in RPMI 1640 supplemented with 10% fetal bovine serum (FBS; 1×106 /mL). H2O2 was added at final concentrations of 0, 125, 250, and 500 µM, and the samples were incubated at 37°C for 4 hour. Alternatively, farnesal (Matrix Scientific, Columbia, SC, USA) was added at final concentrations of 0, 50, 100, 200, and 400 µM, and the samples were incubated similarly. Thereafter, SYTOX Green (Life Technologies, Carlsbad, CA, USA), a membrane-impermeable DNA staining reagent, was added according to the manufacturer’s instructions, and NET-forming neutrophils were detected by flow cytometry (FCM), as described previously.12
NET induction by reaction of VLDL with FC and effects of IMQ and mPSL on VLDL-plus-FC-mediated NET induction
VLDL (100 µg) was reacted with FC (500 µM) and incubated with human peripheral blood neutrophils (1×106/1 mL) at 37°C for 4 hour. IMQ alone (10 µg/mL; Tokyo Chemical Industry, Tokyo, Japan), mPSL alone (10 µM; Tokyo Chemical Industry), or combined IMQ (10 µg/mL) and mPSL (10 µM) was added to VLDL-plus-FC-untreated and VLDL-plus-FC-treated neutrophils, and neutrophils were incubated at 37°C for 4 hour. SYTOX Green was added, and NET-forming neutrophils were detected by FCM. In other experiments, neutrophils (1×106 /mL) seeded into four-well chamber slides (Life Technologies) and settled at 37°C for 30 min were incubated with PBS or 20 nM phorbol myristate acetate (PMA; Sigma-Aldrich) at 37°C for 4 hour. Alternatively, neutrophils were incubated with IMQ (10 µg/mL) and mPSL (10 µM) or IMQ (10 µg/mL), mPSL (10 µM), VLDL (100 µg), and FC (500 µM) at 37°C for 4 hour. After washing with PBS, cells were fixed with 4% PFA for 15 min at RT. The fixed materials were subjected to immunofluorescence staining for NETs. In brief, the samples were first reacted with anti-Cit-H3 antibody (1:100 dilution) and anti-MPO antibody (1:200 dilution; Bio-Rad Laboratories, Hercules, CA, USA) overnight at 4°C. After removal of unbound antibodies using PBS, the samples were next exposed to Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody (1:500 dilution) and Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (1:500 dilution; Sigma-Aldrich) for 1 hour in the dark at RT. Finally, the samples were mounted with a DAPI-containing mounting solution and observed under a fluorescence microscope.
Statistics
Student’s t-test was used to compare two parametric groups. One-way analysis of variance (ANOVA) or Kruskal-Wallis analysis was used to compare multiple parametric or non-parametric groups. p<0.05 was considered statistically significant.
Results
Anti-dsDNA antibody production in IMQ-applied mice
Anti-dsDNA antibody is an outstanding marker of mice with IMQ-induced lupus-like disease.7 The anti-dsDNA antibody titre in day 28 plasma was determined by ELISA. The titre was significantly higher in IMQ-applied mice than in IMQ-unapplied mice (online supplemental figure S1), suggesting that lupus-like disease had developed in IMQ-applied mice.
Supplemental material
Effects of mPSL pulse on histopathology in mice with IMQ-induced lupus-like disease
After confirmation of anti-dsDNA antibody production, IMQ-applied mice were allocated randomly to two groups: Group B without mPSL pulse and group D with mPSL pulse. In the kidneys of group B, some glomeruli showed histopathology compatible with lupus nephritis, such as thickening of the glomerular basement membrane (GBM), PAS-positive hyaline thrombi, and IgG deposition (figure 1A). The proportion of morphologically affected glomeruli exhibiting a thick GBM or PAS-positive hyaline thrombi was significantly higher in group B than in group A (IMQ-unapplied mice without mPSL pulse). mPSL pulse decreased the rate of glomerular impairment in group D, although the trend did not reach statistical significance.
Effects of mPSL pulse on histopathology in mice with IMQ-induced lupus-like disease. Representative (A) renal (PAS) and (B) bone marrow (H&E) findings. (A) A thick GBM and hyaline thrombi are seen in group B and group D. Insets in (A) represent glomerular IgG deposition. Arrowheads in (B) indicate megakaryocytes. Bars, 50 µm. Group A: IMQ-unapplied mice without mPSL pulse (n=6), group B: IMQ-applied mice without mPSL pulse (n=5), group C: IMQ-unapplied mice with mPSL pulse (n=6) and group D: IMQ-applied mice with mPSL pulse (n=6). *p<0.05 (Steel-Dwass comparison in Kruskal-Wallis analysis). GBM, glomerular basement membrane; IMQ, imiquimod; mPSL, methylprednisolone; PAS, periodic acid Schiff.
Megakaryocyte abundance in the bone marrow was significantly greater in group B than in group A (figure 1B). This might have reflected lupus-associated thrombocytopenia, although the number of platelets in the blood was not measured. The number of megakaryocytes in the bone marrow of group D recovered to normal levels, indicating the effect of mPSL pulse.
Ischaemia of the femoral head cartilage in mPSL-administered mice with IMQ-induced lupus-like disease
ONFH development of was examined histopathologically. Because ONFH was not evident in all groups (figure 2A), the ischaemic state of the tissue was assessed by immunohistochemistry (IHC) for HIF-1α, as reported previously.4 Results indicated that IMQ application induced ischaemia of the femoral head cartilage, and mPSL pulse enhanced this effect (figure 2B).
Ischaemia of the femoral head cartilage in mPSL-administered mice with IMQ-induced lupus-like disease. (A) Representative findings of the femoral head (H&E). ONFH was not evident. Bars, 500 µm. (B) To assess the ischaemic state, FFPE sections of the femoral head were subjected to IHC for HIF-1α. The Allred scoring system was used for the semiquantitative evaluation of HIF-1α expression. Bars, 200 µm. Group A: IMQ-unapplied mice without mPSL pulse (n=6), group B: IMQ-applied mice without mPSL pulse (n=5), group C: IMQ-unapplied mice with mPSL pulse (n=6) and group D: IMQ-applied mice with mPSL pulse (n=6). *p<0.05, **p<0.01 (Tukey-Kramer’s comparison in one-way ANOVA). ANOVA, analysis of variance; FFPE, formalin-fixed and paraffin-embedded; HIF-1α, hypoxia-inducible factor 1α; IHC, immunohistochemistry; IMQ, imiquimod; ONFH, idiopathic osteonecrosis of the femoral head; mPSL, methylprednisolone.
Tissue infiltration of NET-forming neutrophils
To demonstrate the infiltration of NET-forming neutrophils around the femoral head, connective tissues containing synovial membrane attached to the femoral head were sampled and subjected to immunofluorescence staining for NETs. Cit-H3-positive, MPO-positive and DAPI-positive NET-forming neutrophils were wedged in small vessels surrounding the femoral head in group D (figure 3A). Corresponding to this, significantly more glomeruli displayed NET deposition in group D than in group B (figure 3B). These findings indicated that NET formation was enhanced in mice with IMQ-induced lupus-like disease after mPSL pulse.
Infiltration of NET-forming neutrophils around the femoral head and NET deposition in glomeruli. (A) Connective tissues attached to the femoral head were sampled, and immunofluorescence staining for NETs was performed. Cit-H3-positive (red), MPO-positive (green) and DAPI-positive (blue) NET-forming neutrophils were wedged in small vessels surrounding the femoral head in group D. Bars, 50 µm. (B) NET deposition in glomeruli. Cit-H3-positive (red), MPO-positive (green) and DAPI-positive (blue) substances indicate NET deposition. Dotted circles indicate glomeruli. Group A: IMQ-unapplied mice without mPSL pulse (n=6), group B: IMQ-applied mice without mPSL pulse (n=5), group C: IMQ-unapplied mice with mPSL pulse (n=6) and group D: IMQ-applied mice with mPSL pulse (n=6). *p<0.05 (Steel-Dwass comparison in Kruskal-Wallis analysis). DAPI, 4′,6-diamidino-2-phenylindole; IMQ, imiquimod; MPO, myeloperoxidase; mPSL, methylprednisolone; NET, neutrophil extracellular trap.
PCYOX1 is associated with enhanced NET formation in mPSL-administered mice with IMQ-induced lupus-like disease
To identify the factors associated with enhanced NET formation in mice with IMQ-induced lupus-like disease that received mPSL pulse, day 56 plasma was pooled in each group and subjected to shotgun proteome analysis. Although the diversity of proteins included was equivalent among samples (figure 4A), expression profiles were generally different between IMQ-applied mice (groups B and D) and IMQ-unapplied mice (groups A and C; figure 4B). Furthermore, some proteins increased exclusively in group D, in which NET formation was enhanced (online supplemental table S1). Among them, PCYOX1 was detected quantitatively in group D but not in group A, B or C (figure 4C). A volcano plot indicated that PCYOX1 was detected in the group D sample but not in the mixed sample of groups A to C (figure 4D). This study focused on PCYOX1 because it has been reported to react with FC as a substrate to produce H2O2,8 9 a known NET inducer.13 H2O2 production by the reaction of PCYOX1 with FC and the dose-dependent NET induction by H2O2 were confirmed (online supplemental figure S2a and S2b).
Supplemental material
Supplemental material
Proteome analysis of plasma. To identify the factors associated with the increased number of NET-forming neutrophils in IMQ-induced lupus mice after mPSL pulse, day 56 plasma pooled per group was subjected to shotgun proteome analysis. (A) Diversity of proteins included in duplicated samples in each group, (B) expression profiling, (C) PCYOX1 expression and (D) a volcano plot, in which PCYOX1 is indicated by a yellow circle. Group A: IMQ-unapplied mice without mPSL pulse (n=6), group B: IMQ-applied mice without mPSL pulse (n=5), group C: IMQ-unapplied mice with mPSL pulse (n=6) and group D: IMQ-applied mice with mPSL pulse (n=6). IMQ, imiquimod; mPSL, methylprednisolone; NET, neutrophil extracellular trap; PCYOX1, prenylcysteine oxidase 1.
In vitro assessment for NET induction by PCYOX1 and its substrate FC
Corresponding to PCYOX1 abundance in VLDL,9 10 reaction of VLDL with FC produced H2O2 in a VLDL dose-dependent manner (figure 5A). Furthermore, farnesal—another product of the reaction of PCYOX1 and FC—induced NETs dose dependently (figure 5B). The combined addition of IMQ and mPSL synergistically enhanced VLDL-plus-FC-induced NET formation, whereas each reagent alone had only minimal effects (figure 5C and D). Parts of these findings were confirmed morphologically (online supplemental figure S3). Based on these findings, it was suggested that PCYOX1 plays a role in NET induction in mice with IMQ-induced lupus-like disease after mPSL pulse.
Supplemental material
In vitro assessment for NET induction by PCYOX1 and its substrate FC. (A) VLDL dose-dependent H2O2 production by reaction with FC. (B) Dose-dependent NET induction by farnesal. Peripheral blood neutrophils were gated as granulocytes in the forward and side scatter profiles and SYTOX Green histograms of gated cells were displayed. R2 gate was set as SYTOX Green-positive neutrophils (NET-forming neutrophils) referring to the histogram of neutrophils without SYTOX Green staining. (C) Combined addition of IMQ and mPSL synergistically enhanced VLDL-plus-FC-induced NET formation (red arrow), whereas each reagent alone had minimal effect. (D) Mean fluorescence intensity (MFI) of SYTOX Green as an indicator of NETs. Experiments were repeated thrice. Data were presented as the mean±SD. *p<0.05 vs PBS (Steel-Dwass comparison in Kruskal-Wallis analysis). ET, neutrophil extracellular trap; FC, farnesylcysteine; IMQ, imiquimod; mPSL, methylprednisolone; PBS, phosphate-buffered saline; PCYOX1, prenylcysteine oxidase 1; VLDL, very low-density lipoproteins.
Discussion
This study demonstrated that mPSL pulse-induced ischaemia of the femoral head cartilage and enhanced NET formation in mice with lupus-like disease. Although ONFH had not yet developed, these findings were consistent with the hypothesis that mPSL pulse for lupus can induce NET formation and result in microcirculation disorders. Because infiltration of NET-forming neutrophils was observed not only in tissues around the femoral head but also in glomeruli, retarded microcirculation occurred systemically in mice with IMQ-induced lupus-like disease after mPSL pulse. Additional factors, such as mechanical stress, may be required for ONFH development.14 Because histones derived from NETs contribute to glomerulonephritis progression,15 NET deposition in glomeruli may be associated with insufficient improvement in mice with IMQ-induced lupus-like disease after mPSL pulse.
We could not determine anti-dsDNA antibody titres in day 56 plasma because the samples were used for proteome analysis. In addition, it remains elusive whether NETs were deposited in the femoral head tissue because non-specific staining occurred in NET staining, probably due to decalcification necessary for bone tissue processing. These are the limitations of this study.
Proteome analysis of the plasma suggested that PCYOX1 abundance, mainly in VLDL, contributed to mPSL pulse-mediated NET induction. In preliminary IHC using FFPE liver sections (data not shown), PCYOX1-positive findings were observed in group B (IMQ-applied mice without mPSL administration) and group D (IMQ-applied mice with mPSL administration). PCYOX1 was evident on the surface of lipids deposited in hepatocytes. In contrast, PCYOX1 in the plasma was detected exclusively in group D mice (see figure 4C,D). Therefore, it was hypothesised that IMQ can induce PCYOX1 expression in the liver and mPSL pulse can recruit PCYOX1 into the plasma, consistent with the literature reporting that glucocorticoids induce hyperlipidaemia16; however, further studies are needed to confirm this. Although plasma VLDL levels were not evaluated in this study, they were reportedly higher in SLE patients than in healthy subjects.17 18 Moreover, glucocorticoid administration to SLE patients worsened lipid abnormalities.19 Therefore, it is likely that mPSL pulse for lupus can increase plasma VLDL levels, increasing PCYOX1 levels.
PCYOX1 reacts with FC as a substrate to produce H2O2 and farnesal.8 FC is a component of cell membranes.9 PCYOX1 in VLDL accumulates on the vascular endothelial surface of large vessels, reacting with FC to produce H2O2 and farnesal. This study demonstrated that not only H2O2 but also farnesal can induce NETs. Neutrophils attached to the vascular endothelium are likely exposed to H2O2 and farnesal and then release NETs.
Interestingly, the combined addition of IMQ and mPSL synergistically enhanced VLDL-plus-FC-induced NET formation, whereas each reagent alone had minimal effects. One possible explanation is that the combined addition of IMQ and mPSL may raise the ability of neutrophils to generate NETs on H2O2 and farnesal exposure. Further studies are needed to uncover the precise mechanism.
Conclusion
Although this study focused on the adverse aspects of mPSL pulse therapy, it did not intend to demean the clinical importance of this treatment. This study suggested that mPSL pulse for lupus can induce NET formation, resulting in microcirculation disorders. Related factors, including PCYOX1, VLDL, H2O2 and farnesal, are worthy of attention to understand the underlying mechanisms and create novel therapeutic strategies for mPSL-mediated microcirculation disorders, including ONFH.
Data availability statement
Data are available on reasonable request.
Ethics statements
Patient consent for publication
Ethics approval
Experiments using human materials were permitted by the Ethics Committee of the Faculty of Health Sciences, Hokkaido University (approval number 20-40) and performed in accordance with the Declaration of Helsinki. After obtaining written informed consent, peripheral blood (10 ml) was drawn from healthy volunteers. Participants gave informed consent to participate in the study before taking part.
Acknowledgments
We thank the animal facility staff at the Graduate School of Medicine, Hokkaido University for breeding mice.
Supplementary materials
Supplementary Data
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
Footnotes
Contributors HO, SY, YH, AS, NO, RY, TS, IN, SA, MT, YN, SM and AI performed the experiments. HO, SY, RY, TS, YN, SM, DN, UT, NI and AI analysed the data. HO, TS, YN, SM and AI designed the study. HO, TS, YN, SM, DN, UT and AI wrote the manuscript. All authors read the final version of the manuscript and provided critical feedback. AI is the author acting as guarantor.
Funding This work was supported by the Japanese Grant-in-Aid for Scientific Research (numbers 20K17984 and 23K08689 for TS).
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.