C5a-C5aR1 axis controls mitochondrial fission to promote podocyte injury in lupus nephritis.

Podocytes are essential to maintaining the integrity of the glomerular filtration barrier, but they are frequently affected in lupus nephritis (LN). Here, we show that the significant upregulation of Drp1S616 phosphorylation in podocytes promotes mitochondrial fission, leading to mitochondrial dysfunction and podocyte injury in LN. Inhibition or knockdown of Drp1 promotes mitochondrial fusion and protects podocytes from injury induced by LN serum. In vivo, pharmacological inhibition of Drp1 reduces the phosphorylation of Drp1S616 in podocytes in lupus-prone mice. Podocyte injury is reversed when Drp1 is inhibited, resulting in the alleviation of proteinuria. Mechanistically, complement component C5a (C5a) upregulates the phosphorylation of Drp1S616 and promotes mitochondrial fission in podocytes. Moreover, the expression of C5a receptor 1 (C5aR1) is notably upregulated in podocytes in LN. C5a-C5aR1 axis-controlled phosphorylation of Drp1S616 and mitochondrial fission are substantially suppressed when C5aR1 is knocked down by siRNA. Moreover, lupus-prone mice treated with C5aR inhibitor show reduced phosphorylation of Drp1S616 in podocytes, resulting in significantly less podocyte damage. Together, this study uncovers a novel mechanism by which the C5a-C5aR1 axis promotes podocyte injury by enhancing Drp1-mediated mitochondrial fission, which could have significant implications for the treatment of LN.

Spleen fibroblastic reticular cell-derived acetylcholine promotes lipid metabolism to drive autoreactive B cell responses.

Autoreactive B cell responses are essential for the development of systemic lupus erythematosus (SLE). Fibroblastic reticular cells (FRCs) are known to construct lymphoid compartments and regulate immune functions. Here, we identify spleen FRC-derived acetylcholine (ACh) as a key factor that controls autoreactive B cell responses in SLE. In SLE, CD36-mediated lipid uptake leads to enhanced mitochondrial oxidative phosphorylation in B cells. Accordingly, the inhibition of fatty acid oxidation results in reduced autoreactive B cell responses and ameliorated diseases in lupus mice. Ablation of CD36 in B cells impairs lipid uptake and differentiation of autoreactive B cells during autoimmune induction. Mechanistically, spleen FRC-derived ACh promotes lipid influx and generation of autoreactive B cells through CD36. Together, our data uncover a novel function of spleen FRCs in lipid metabolism and B cell differentiation, placing spleen FRC-derived ACh in a key position in promoting autoreactive B cells in SLE.

NAMPT is a metabolic checkpoint of IFNγ-producing CD4+ T cells in lupus nephritis.

Interferon γ (IFNγ) produced by T cells represents the featured cytokine and is central to the pathogenesis of lupus nephritis (LN). Here, we identified nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the salvage NAD+ biosynthetic pathway, as playing a key role in controlling IFNγ production by CD4+ T cells in LN. Our data revealed that CD4+ T cells from LN showed an enhanced NAMPT-mediated NAD+ biosynthetic process, which was positively correlated with IFNγ production in CD4+ T cells. NAMPT promoted aerobic glycolysis and mitochondrial respiration in CD4+ T cells from patients with LN or MRL/lpr mice through the production of NAD+. By orchestrating metabolic fitness, NAMPT promoted translational efficiency of Ifng in CD4+ T cells. In vivo, knockdown of NAMPT by small interfering RNA (siRNA) or pharmacological inhibition of NAMPT by FK866 suppressed IFNγ production in CD4+ T cells, leading to reduced inflammatory infiltrates and ameliorated kidney damage in lupus mice. Taken together, this study uncovers a metabolic checkpoint of IFNγ-producing CD4+ T cells in LN in which therapeutically targeting NAMPT has the potential to normalize metabolic competence and blunt pathogenicity of CD4+ T cells in LN.

Activation of necroptosis pathway in podocyte contributes to the pathogenesis of focal segmental glomerular sclerosis.

BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is characterized by podocyte damage and severe proteinuria. The exact mechanism of podocyte damage and loss remains unclear. Necroptosis, a lytic form of programmed cell death mediated by RIP3 and MLKL, has emerged as an important cell death pattern in multiple tissues and cell types. Necroptosis in FSGS has not been investigated. METHODS: Public GEO data regarding podocyte treated with vehicle or adriamycin (ADR) was identified and analyzed. Cultured human podocytes were used to explore the activation of necroptosis upon ADR stimulation. The expression of necroptosis pathway molecules, p-RIP3 and p-MLKL, was examined in the glomeruli and defoliated urinary podocytes of patients with FSGS. The effect of necroptosis inhibition was assessed in ADR-induced glomerulopathy mice using GSK872. RESULTS: Publicly available RNA-sequencing data analysis showed that both necroptosis and NLRP3 inflammasome pathway were up-regulated in ADR-injured podocyte. Immunofluorescent staining showed increased expression of p-RIP3 and p-MLKL, the active forms of RIP3 and MLKL, in podocytes of FSGS patients and ADR-induced glomerulopathy mice but not in the normal control. GSK872, an RIP3 kinase inhibitor, significantly inhibited the expression of p-RIP3, p-MLKL and activation of NLRP3 in cultured podocytes treated with ADR. GSK872 treatment of mice with ADR-induced nephropathy resulted in the reduced expression of p-RIP3, p-MLKL, NLRP3 and caspase-1 p20. GSK872 also significantly inhibited the expression of p-MLKL in the podocytes of ADR-induced nephropathy, resulting in the attenuation of proteinuria and renal histological lesions. CONCLUSION: Necroptosis pathway might be a valuable target for the treatment of FSGS.

Iron controls T helper cell pathogenicity by promoting glucose metabolism in autoimmune myopathy.

BACKGROUND: T helper cells in patients with autoimmune disease of idiopathic inflammatory myopathies (IIM) are characterized with the proinflammatory phenotypes. The underlying mechanisms remain unknown. METHODS: RNA sequencing was performed for differential expression genes. Gene expression in CD4+ T-cells was confirmed by quantitative real-time PCR. CD4+ T-cells from IIM patients or healthy controls were evaluated for metabolic activities by Seahorse assay. Glucose uptake, T-cell proliferation and differentiation were evaluated and measured by flow cytometry. Human CD4+ T-cells treated with iron chelators or Pfkfb4 siRNA were measured for glucose metabolism, proliferation and differentiation. Signalling pathway activation was evaluated by western blot and flow cytometry. Mouse model of experimental autoimmune myositis (EAM) were induced and treated with iron chelator or rapamycin. CD4+ T-cell differentiation and muscle inflammation in the EAM mice were evaluated. RESULTS: RNA-sequencing analysis revealed that iron was involved with glucose metabolism and CD4+ T-cell differentiation. IIM patient-derived CD4+ T-cells showed enhanced glycolysis and mitochondrial respiration, which was inhibited by iron chelation. CD4+ T-cells from patients with IIM was proinflammatory and iron chelation suppressed the differentiation of interferon gamma (IFNγ)- and interleukin (IL)-17A-producing CD4+ T-cells, which resulted in an increased percentage of regulatory T (Treg) cells. Mechanistically, iron promoted glucose metabolism by an upregulation of PFKFB4 through AKT-mTOR signalling pathway. Notably, the knockdown of Pfkfb4 decreased glucose influx and thus suppressed the differentiation of IFNγ- and IL-17A-producing CD4+ T-cells. In vivo, iron chelation inhibited mTOR signalling pathway and reduced PFKFB4 expression in CD4+ T-cells, resulting in reduced proinflammatory IFNγ- and IL-17A-producing CD4+ T-cells and increased Foxp3+ Treg cells, leading to ameliorated muscle inflammation. CONCLUSIONS: Iron directs CD4+ T-cells into a proinflammatory phenotype by enhancing glucose metabolism. Therapeutic targeting of iron metabolism should have the potential to normalize glucose metabolism in CD4+ T-cells and reverse their proinflammatory phenotype in IIM.

CaMK4 Promotes Acute Lung Injury Through NLRP3 Inflammasome Activation in Type II Alveolar Epithelial Cell.

Background: Type II alveolar epithelial cell (AEC II), in addition to its roles in maintaining lung homeostasis, takes an active role in inflammatory response during acute lung injury (ALI). Ca2+/calmodulin-dependent protein kinase IV (CaMK4) activated by Ca2+/calmodulin signaling, has been implicated in immune responses. This study was to investigate the roles of CaMK4 in the development of ALI and the underlying mechanisms. Methods: CaMK4 inhibitor KN-93 was used to investigate the effects of CaMK4 on NLRP3 inflammasome activation. The effects of KN-93 on disease development of lipopolysaccharide (LPS)-induced ALI were also evaluated. The role of CaMK4 on NLRP3 inflammasome activation was explored in human AEC II cell line A549 using KN-93 or CaMK4 siRNA. NLRP3 inflammasome activation was measured by histology immunofluorescence and Western blot. IL-1ß and IL-18 were measured by ELISA. Results: Phosphorylation of CaMK4 and the expression of NLRP3 and Caspase-1 p20 were increased in the lungs of LPS-induced ALI mice, which was suppressed by KN-93 as measured by Western blot. Further, the activation of NLRP3 inflammasome was detected in AEC II from patients with acute respiratory distress syndrome (ARDS) and LPS-induced ALI mice. In vitro, inhibition or silencing CaMK4 in AEC II significantly inhibited NLRP3 inflammasome activation, resulting in reduced IL-1ß production. The inhibition of NLRP3 inflammasome and decreased IL-1ß/IL-18 production by KN-93 led to reduced inflammatory infiltration and ameliorated lung injury in LPS-induced ALI mice. Conclusion: CaMK4 controls the activation of NLRP3 inflammasome in AEC II during LPS-induced ALI. CaMK4 inhibition could be a novel therapeutic approach for the treatment of ALI.

Tfh cells with NLRP3 inflammasome activation are essential for high-affinity antibody generation, germinal centre formation and autoimmunity.

OBJECTIVE: NLRP3 inflammasome regulates T cell responses. This study examined the roles of NLRP3 inflammasome activation in the regulation of T follicular helper (Tfh) cells during humoral response to T dependent antigens and in systemic lupus erythematosus (SLE). METHODS: NLRP3 inflammasome activation of Tfh cells was studied in B6, MRL/lpr and NZM2328 mice and in SLE patients and healthy controls using a fluorescence-labelled caspase-1 inhibitor probe. MCC950, a selective inhibitor of NLRP3, was used to investigate the relation between NLRP3 inflammasome activation and germinal centre (GC) reaction, Ab responses to immunisation, and autoantibody production. RESULTS: NLRP3 inflammasome activation in Tfh cells after immunisation was identified in B6 mice. MCC950 inhibited humoral responses to sheep red blood cell and NP-CGG with reduction of the GC reaction. B6 mice with lymphoid cell-specific deletion of NLRP3 or Casp1 mounted suboptimal humoral responses with impaired GC formation and defective affinity maturation. In MRL/lpr and NZM2328 mice, inhibition of NLRP3 activation suppressed NLRP3 activated Tfh cell expansion as well as attenuated lupus-like phenotypes. Tfh cells with activated NLRP3 inflammasome exhibited increased expression of molecules for Tfh cell function and differentiation, and had greater ability to activate B cells. In SLE patients, disease activity was positively correlated with an increase in the activated NLRP3+ Tfh population and this population was markedly reduced in response to therapy. CONCLUSIONS: The activation of NLRP3 inflammasome in Tfh cells is an integral part of responses to immunisation. The activated NLRP3+ Tfh population is essential for optimal humoral responses, GC formation and autoimmunity.

CRAC Channel Controls the Differentiation of Pathogenic B Cells in Lupus Nephritis.

Store-operated Ca2+ release-activated Ca2+ (CRAC) channel is the main Ca2+ influx pathway in lymphocytes and is essential for immune response. Lupus nephritis (LN) is an autoimmune disease characterized by the production of autoantibodies due to widespread loss of immune tolerance. In this study, RNA-seq analysis revealed that calcium transmembrane transport and calcium channel activity were enhanced in naive B cells from patients with LN. The increased expression of ORAI1, ORAI2, and STIM2 in naive B cells from patients with LN was confirmed by flow cytometry and Western blot, implying a role of CRAC channel in B-cell dysregulation in LN. For in vitro study, CRAC channel inhibition by YM-58483 or downregulation by ORAI1-specific small-interfering RNA (siRNA) decreased the phosphorylation of Ca2+/calmodulin-dependent protein kinase2 (CaMK2) and suppressed Blimp-1 expression in primary human B cells, resulting in decreased B-cell differentiation and immunoglobulin G (IgG) production. B cells treated with CaMK2-specific siRNA showed defects in plasma cell differentiation and IgG production. For in vivo study, YM-58483 not only ameliorated the progression of LN but also prevented the development of LN. MRL/lpr lupus mice treated with YM-58483 showed lower percentage of plasma cells in the spleen and reduced concentration of anti-double-stranded DNA antibodies in the sera significantly. Importantly, mice treated with YM-58483 showed decreased immune deposition in the glomeruli and alleviated kidney damage, which was further confirmed in NZM2328 lupus mice. Collectively, CRAC channel controlled the differentiation of pathogenic B cells and promoted the progression of LN. This study provides insights into the pathogenic mechanisms of LN and that CRAC channel could serve as a potential therapeutic target for LN.

Circulating TFH cells is correlated with disease activity in anti-MDA5 antibody positive idiopathic inflammatory myopathies.

OBJECTIVES: Idiopathic inflammatory myopathies (IIM) are a group of disorders characterised by the production of autoantibodies and inflammatory infiltrates in the skeletal muscles. Follicular T helper (TFH) cells are known to be crucial for B cell differentiation and autoantibody production in autoimmune diseases. The aim of this study was to investigate the involvement of TFH cells in IIM. METHODS: Circulating TFH cells in 44 IIM patients or 11 age- and gender-matched healthy controls (HCs) were measured by flow cytometry. ICOS, PD-1, active caspase-1 and Ki-67 expression in TFH cells was examined. The correlations between the frequency of TFH cells and clinical disease activities were also analysed. RESULTS: The frequency of TFH cells was 16.6% in IIM patients with anti-melanoma differentiation-associated gene (MDA5) antibody compared to 10.6% and 12.9% in anti-MDA5 negative patients or HCs, respectively (both p<0.05). The frequency of TFH cells was positively correlated with clinical disease activities: patient/parent's assessment VAS (r=0.51, p<0.05), physician's assessment VAS (r=0.59, p<0.05) and MYOACT scores (total systems: r=0.62, p<0.05; extramuscular system: r=0.56, p<0.05; pulmonary system, r=0.55, p<0.05). The percentage of PD-1highICOShigh TFH cells was 3.68% in anti-MDA5 positive patients compared to 2.70% and 1.96% in anti-MDA5 negative patients or HCs, respectively (both p<0.05). The percentage of Ki-67 positive TFH cells was 3.50% in anti-MDA5 positive patients compared to 2.36% and 1.76% in anti-MDA5 negative patients or HCs, respectively (p<0.05). Interestingly, active caspase-1 was significantly increased in TFH cells in anti-MDA5 positive patients compared to the patients without anti-MDA5 or HCs (3.30% vs. 1.67% and 3.30% vs. 1.02%, both p<0.001). CONCLUSIONS: These data suggest a role for TFH cells in the pathogenesis of anti-MDA5 positive IIM and TFH cells might serve as a disease biomarker for this subset of patients.

JAK/STAT signaling controls the fate of CD8+CD103+ tissue-resident memory T cell in lupus nephritis.

Autoimmune mediated inflammation and renal damage in lupus nephritis (LN) depends partly on the infiltration of lymphocytes in glomeruli and renal interstitium. Here we identified a population of CD8+ T cells with a CD103+-phenotype in the healthy kidneys of human and mouse. These cells were typically CD69+CD103+ tissue-resident memory T cells (TRM) in the kidney. CD8+ TRM cells were expanded in the kidneys of patients with LN or MRL/lpr mice. The expansion of renal CD8+ TRM cells correlated significantly with kidney disease activity. These cells were active in producing cytokines, perforin and granzyme B in the kidney of MRL/lpr mice. Importantly, renal CD8+ TRM cells underwent proliferation and self-renewal to maintain a stable TRM pool in the kidney of MRL/lpr mice, contributing to renal inflammation and damage. JAK/STAT signaling in the MRL/lpr mice was required for renal TRM self-renewal as well as maintenance of effector functions. Targeting JAK/STAT signaling by tofacitinib effectively suppressed effector functions and impaired the survival of renal TRM cells in the kidney, contributing to improved kidney function in MRL/lpr mice. These results provided evidences that renal CD8+ TRM cells play a role in the pathogenesis of LN. They could serve as a therapeutic target for LN.

Pathogenesis of lupus nephritis: RIP3 dependent necroptosis and NLRP3 inflammasome activation.

RIP3 activation leads to activation of necroptosis and the NLRP3 inflammasome pathways. The activation of RIP3 in lupus nephritis (LN) has not been investigated. In this study, RIP3 and necroptosis pathway activations were demonstrated in podocytes in renal biopsies from patients with class IV LN and in the diseased kidneys from lupus-prone NZM2328 and MRL/lpr mice. RIP3 activation was accompanied with the activation of MLKL, the effector molecule of the necroptosis pathway, and activation of caspase-1, the effector of the NLRP3 inflammasome pathway. Podocyte activation of RIP3 was detected readily with the development of LN in NZM2328 mice, suggesting this activation may play a significant role in the pathogenesis of LN. GSK872, a RIP3 specific inhibitor, inhibited the development of LN in MRL/lpr mice with down-regulation of RIP3 activation in podocytes, decreased the splenic sizes and weights and anti-dsDNA antibody titers. IgG from pooled sera of diseased NZM2328 mice succumbing to LN induced both the necroptosis pathway and NLRP3 inflammasome activation in a podocyte cell line and this activation was specifically blocked by GSK872. These results indicate that the necroptosis pathway and the RIP3 dependent NLRP3 inflammasome pathway are activated in podocytes during LN. Inhibition of RIP3 kinase may be a novel therapeutic approach to treat LN and systemic lupus erythematosus (SLE).

Innate lymphoid cell disturbance with increase in ILC1 in systemic lupus erythematosus.

The innate lymphoid cell (ILC) is a group of effector cells with diverse important cellular functions in both health and disease states. In comparison with healthy controls, there were increases in circulating ILC in SLE patients. The proportion of ILC1 significantly increased with significant decreases of ILC2 in SLE patients and ILC3 in SLE patients with moderate to severe activity. IL-12, IL-18, IL-25, IL-33, IL-23, IL-1ß and IFN-γ were significantly increased in SLE patients. Moreover, IL-12, IL-18 and IL-1ß but not IFN-γ correlated significantly with SLEDAI. Successful treatments rapidly reduced them and with certain normalization of the ILC subsets. In addition to increases in ILC1 numbers, ~ 80% of the ILC1 in SLE patients were positive for synthesis of IFN-γ. Plasma from SLE patients were shown to be potent in inducing ILC1. Thus, increased circulating ILC1 might contribute to the pathogenesis of SLE through mounting type 1 immune response.

Pim-1 as a Therapeutic Target in Lupus Nephritis.

OBJECTIVE: Lupus nephritis (LN) is a major determinant of morbidity and mortality in systemic lupus erythematosus (SLE). Pim-1 regulates lymphocyte proliferation and activation. The role of Pim-1 in autoimmune disease remains unclear. This study was undertaken to test the hypothesis that inhibition of Pim-1 would have therapeutic potential in patients with LN. METHODS: Pim-1 expression was analyzed in lupus-prone (NZB × NZW)F1 mice (n = 6), human peripheral blood mononuclear cells (PBMCs) from SLE patients (n = 10), and glomeruli from patients with LN (n = 8). The therapeutic effect of the Pim-1 inhibitor AZD1208 was assessed in the same murine lupus model (n = 10 mice per group). In vitro analysis was conducted to explore the mechanisms of action of Pim-1 in mouse and human podocytes after Pim-1 expression had been induced by anti-double-stranded DNA (anti-dsDNA) antibody-positive serum. Finally, MRL/lpr mice were used to confirm the therapeutic effects of Pim-1 inhibition in vivo (n = 10 mice per group). RESULTS: Up-regulation of Pim-1 was seen in renal lysates from diseased (NZB × NZW)F1 mice and in PBMCs from patients with SLE and renal biopsy tissue from patients with LN, relative to their control counterparts (each P < 0.05). The Pim-1 inhibitor AZD1208 reduced the severity of proteinuria, glomerulonephritis, renal immune complex deposits, and serum anti-dsDNA antibody levels, concomitant with the suppression of NFATc1 expression and NLRP3 inflammasome activation, in diseased (NZB × NZW)F1 mice (each P < 0.05 versus controls). Moreover, in mouse and human podocytes, Pim-1 knockdown with targeted small interfering RNA (siRNA) suppressed NFATc1 and NLRP3 inflammasome signaling in the presence of anti-dsDNA-positive serum (each P < 0.05 versus control siRNA). Mechanistically, Pim-1 modulated NLRP3 inflammasome activation through intracellular Ca2+ (P < 0.05 versus normal controls). The therapeutic effect of Pim-1 blockade was replicated in MRL/lpr mice. CONCLUSION: These data identify Pim-1 as a critical regulator of LN pathogenesis in patients with SLE. Targeting of the Pim-1/NFATc1/NLRP3 pathway might therefore have therapeutic potential in human LN.

The ratio of circulating follicular T helper cell to follicular T regulatory cell is correlated with disease activity in systemic lupus erythematosus.

Follicular T regulatory (Tfr) cells inhibit follicular T helper (Tfh) cells mediated B cell responses. Tfh cells are involved in the pathogenesis of systemic lupus erythematosus (SLE). However, the role of Tfr cells in SLE remains unclear. The frequency of circulating Tfr and Tfh cells were examined in SLE patients and healthy controls. The frequency of circulating Tfr cell decreased and Tfh/Tfr ratio increased in SLE patients. Serum anti-dsDNA antibody level positively correlated with frequency of Tfh cells and Tfh/Tfr ratios but negatively correlated with the frequency of Tfr cells. Moreover, the frequency of Tfr and Tfh/Tfr ratio but not that of Tfh was correlated with diseases activity. In addition, increase in Tfr cell numbers and decrease in the Tfh/Tfr ratios were observed with successful treatments. Thus, Tfr cells should be considered as a biomarker for SLE and their role in the pathogenesis of SLE warrants further investigation.

Podocyte Activation of NLRP3 Inflammasomes Contributes to the Development of Proteinuria in Lupus Nephritis.

OBJECTIVE: Development of proteinuria in lupus nephritis (LN) is associated with podocyte dysfunction. The NLRP3 inflammasome has been implicated in the pathogenesis of LN. The purpose of this study was to investigate whether NLRP3 inflammasome activation is involved in the development of podocyte injury in LN. METHODS: A fluorescence-labeled caspase 1 inhibitor probe was used to detect the activation of NLRP3 inflammasomes in podocytes derived from lupus-prone NZM2328 mice and from renal biopsy tissues obtained from patients with LN. MCC950, a selective inhibitor of NLRP3, was used to treat NZM2328 mice. Proteinuria, podocyte ultrastructure, and renal pathology were evaluated. In vitro, sera from diseased NZM2328 mice were used to stimulate a podocyte cell line, and the cells were analyzed by flow cytometry. RESULTS: NLRP3 inflammasomes were activated in podocytes from lupus-prone mice and from patients with LN. Inhibition of NLRP3 with MCC950 ameliorated proteinuria, renal histologic lesions, and podocyte foot process effacement in lupus-prone mice. In vitro, sera from diseased NZM2328 mice activated NLRP3 inflammasomes in the podocyte cell line through the production of reactive oxygen species. CONCLUSION: NLRP3 inflammasomes were activated in podocytes from lupus-prone mice and from LN patients. Activation of NLRP3 is involved in the pathogenesis of podocyte injuries and the development of proteinuria in LN.

Anti-dsDNA antibodies bind to TLR4 and activate NLRP3 inflammasome in lupus monocytes/macrophages.

BACKGROUND: NLRP3 inflammasome has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). The activation of NLRP3 inflammasome results in the production of IL-1ß and the subsequent inflammation. Anti-dsDNA antibodies (anti-dsDNA Abs) play critical roles in the development and progression of SLE. However, the mechanism of NLRP3 inflammasome activation in SLE is still not known. This study investigated the activation of NLRP3 inflammasome stimulated by anti-dsDNA Abs in monocytes/macrophages from SLE patients. METHODS: Monocytes/macrophages from SLE patients or healthy controls were stimulated with anti-dsDNA Ab-positive serum or purified anti-dsDNA Abs. Activation of inflammasome was measured by flow cytometry or Western blot. Anti-dsDNA Abs isolated from active SLE patients were injected into female (NZB × NZW) F1 mice and the activation of NLRP3 inflammasome and the frequencies of Th17 and Treg were examined. RESULTS: The activity of caspase-1 was significantly increased in active SLE patients and was correlated with serum levels of anti-dsDNA Abs and disease activities. The concentrations of IL-1ß and IL-17A were also significantly higher in SLE patients compared to healthy controls. Anti-dsDNA Ab-positive serum rather than healthy serum or RF (rheumatoid factor)-positive serum stimulated the activation of caspase-1 in monocytes. Anti-dsDNA Abs bound to TLR4 on macrophages and induced the production of ROS. Mitochondria-targeting antioxidant Mito-TEMPO, IκB kinase inhibitor peptide or TLR4 siRNA inhibited the activation of NLRP3 inflammasome and the secretion of IL-1ß induced by anti-dsDNA Abs. Injection of anti-dsDNA Abs into (NZB × NZW) F1 mice resulted in increased caspase-1 activation and production of IL-1ß and IL-17A. The Th17/Treg cell ratio also significantly increased following anti-dsDNA Ab injection. CONCLUSIONS: Anti-dsDNA Abs activated NLRP3 inflammasome in monocytes/macrophages from SLE patients by binding to TLR4 and inducing the production of mitochondrial ROS.

Myeloid-derived suppressor cells contribute to bone erosion in collagen-induced arthritis by differentiating to osteoclasts.

Bone erosion is a sign of severe rheumatoid arthritis and osteoclasts play a major role in the bone resorption. Recently, myeloid-derived suppressor cells (MDSC) has been reported to be increased in collagen-induced arthritis (CIA). The number of circulating MDSCs is shown to correlate with rheumatoid arthritis. These findings suggest that MDSCs are precursor cells involved in bone erosion. In this study, MDSCs isolated from mice with CIA stimulated with M-CSF and RANKL in vitro expressed osteoclast markers and acquired osteoclast bone resorption function. MDSCs sorted from CIA mice were transferred into the tibia of normal DBA/1J mice and bones were subjected to histological and Micro CT analyses. The transferred CIA-MDSCs were shown to differentiate into TRAP(+) osteoclasts that were capable of bone resorption in vivo. MDSCs isolated from normal mice had more potent suppressor activity and much less capability to differentiate to osteoclast. Additional experiments showed that NF-κB inhibitor Bay 11-7082 or IκB inhibitor peptide blocked the differentiation of MDSCs to osteoclast and bone resorption. IL-1Ra also blocked this differentiation. In contrast, the addition of IL-1α further enhanced osteoclast differentiation and bone resorption. These results suggest that MDSCs are a source of osteoclast precursors and inflammatory cytokines such as IL-1, contributing significantly to erosive changes seen in rheumatoid arthritis and related disorders.