Activated Lymphocyte-derived DNA Research Articles

Activated Lymphocyte-Derived DNA Drives Glucose Metabolic Adaptation for Inducing Macrophage Inflammatory Response in Systemic Lupus Erythematosus.

Activated lymphocyte-derived DNA (ALD-DNA) has been reported to drive the polarization of macrophages toward M2b, producing inflammatory cytokines and inducing inflammation, correspondingly playing an essential role in the development of systemic lupus erythematosus (SLE). Recently, accumulating evidence has pinpointed metabolic adaptation as the crucial cell-intrinsic determinant for inflammatory response, in which glucose metabolism is the key event. However, whether and how glucose metabolism was involved in ALD-DNA-induced macrophage inflammatory response and SLE development remains unclear. Herein, we performed glucose metabolomic analyses of ALD-DNA-stimulated macrophages and uncovered increased glycolysis and diminished pentose phosphate pathway (PPP), as well as enhanced glycogenesis. In ALD-DNA-stimulated macrophages, increased glycolysis resulted in higher lactate production, whereas diminished PPP efficiently led to lower levels of nicotinamide adenine dinucleotide phosphate (NADPH) with higher levels of reactive oxygen species (ROS). While blockade of lactate generation exerted no significant effect on macrophage inflammation in response to ALD-DNA, scavenging ROS fundamentally inhibited the inflammatory response of ALD-DNA-stimulated macrophages. Further, cyclic adenosine monophosphate (cAMP), a master for regulating glycogen metabolism, was downregulated by ALD-DNA in macrophages, which subsequently imbalanced glycogen metabolism toward glycogenesis but not glycogenolysis. Administration of cAMP effectively restored glycogenolysis and enhanced PPP, which correspondingly reduced ROS levels and inhibited the inflammatory response of ALD-DNA-stimulated macrophages. Finally, blocking glucose metabolism using 2-deoxy-D-glucose (2-DG) efficiently restricted macrophage inflammatory response and alleviated ALD-DNA-induced lupus disease. Together, our findings demonstrate that ALD-DNA drives the adaptation of glucose metabolism for inducing macrophage inflammatory response in SLE, which might further our understanding of disease pathogenesis and provide clues for interventive explorations.

Extracellular HMGB1 augments macrophage inflammation by facilitating the endosomal accumulation of ALD-DNA via TLR2/4-mediated endocytosis

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with unclear pathogenesis. We previously reported that syngenetic, activated lymphocyte-derived DNA (ALD-DNA) could robustly elicit macrophage activation, which plays an important role in the pathogenesis of murine lupus nephritis. In addition, extracellular HMGB1 obviously facilitated the accumulation of ALD-DNA in endosomes and promoted macrophage inflammation. While the detailed mechanism was still unknown. In this study, we found that HMGB1 could obviously change the DNA uptake pathways in macrophages. ALD-DNA alone was mainly uptake by the low efficient and unselective macropinocytosis, while extracellular HMGB1 potently promoted the more efficient and specific clathrin-/caveolin-1-dependent receptor-mediated endocytosis pathways, and led to the rapid and abundant aggregation of ALD-DNA in endosomes. This effect relied on the DNA binding ability and TLR2/TLR4 of HMGB1. Our study not only helped us to understand the promotion mechanisms of extracellular HMGB1 on ALD-DNA-induced macrophage inflammation, but also provided some clues to the pathogenesis of SLE.

High salt diet accelerates the progression of murine lupus through dendritic cells via the p38 MAPK and STAT1 signaling pathways

The increased incidence of systemic lupus erythematosus (SLE) in recent decades might be related to changes in modern dietary habits. Since sodium chloride (NaCl) promotes pathogenic T cell responses, we hypothesize that excessive salt intake contributes to the increased incidence of autoimmune diseases, including SLE. Given the importance of dendritic cells (DCs) in the pathogenesis of SLE, we explored the influence of an excessive sodium chloride diet on DCs in a murine SLE model. We used an induced lupus model in which bone marrow-derived dendritic cells (BMDCs) were incubated with activated lymphocyte-derived DNA (ALD-DNA) and transferred into C57BL/6 recipient mice. We observed that a high-salt diet (HSD) markedly exacerbated lupus progression, which was accompanied by increased DC activation. NaCl treatment also stimulated the maturation, activation and antigen-presenting ability of DCs in vitro. Pretreatment of BMDCs with NaCl also exacerbated BMDC-ALD-DNA-induced lupus. These mice had increased production of autoantibodies and proinflammatory cytokines, more pronounced splenomegaly and lymphadenopathy, and enhanced pathological renal lesions. The p38 MAPK–STAT1 pathway played an important role in NaCl-induced DC immune activities. Taken together, our results demonstrate that HSD intake promotes immune activation of DCs through the p38 MAPK–STAT1 signaling pathway and exacerbates the features of SLE. Thus, changes in diet may provide a novel strategy for the prevention or amelioration of lupus or other autoimmune diseases.

Administration of activated lymphocyte-derived DNA accelerates and aggravates lupus nephritis in B6/lpr mice: a new approach to modify a lupus murine model.

B6/lpr mouse strain is a well-known systemic lupus erythematosus murine model characterized by uncontrolled lymphoproliferation and autoantibody production. However, it displays a delayed and mild development of lupus nephritis (LN), which is not conducive to the research of the pathogenesis and therapeutic strategies of this condition. Our previous study demonstrated that activated lymphocyte-derived DNA (ALD-DNA) could induce high urine protein levels and severe glomerulonephritis (GN) in BALB/c mice. In the present study, we tried to remedy delayed urine protein production and mild GN in B6/lpr mice via ALD-DNA immunization. We found that urine protein levels were enhanced significantly in B6/lpr mice 4weeks after ALD-DNA immunization compared with those in unactivated lymphocyte-derived (UnALD)-DNA- and phosphate-buffered saline (PBS)-treated controls. Moreover, more serious GN and glomerular immune complex were observed in ALD-DNA-immunized B6/lpr mice. We further explored the mechanism, and found that ALD-DNA immunization promoted T helper type 17 (Th17) cell enrichment remarkably, which enhanced the proportion of autoantibody-secreting plasma cells and promoted the production of anti-dsDNA autoantibodies, leading to accelerated and aggravated LN. Our data demonstrated that ALD-DNA immunization could remedy delayed urine protein production and mild GN in B6/lpr mouse, which makes it more suitable for studies on the pathogenesis of and therapeutic strategies against LN.

TIPE2 Alleviates Systemic Lupus Erythematosus Through Regulating Macrophage Polarization

Background/Aims: We have recently shown that macrophage polarization may alter the pathogenesis and severity of systemic lupus erythematosus (SLE). However, a practical approach to modulate macrophage polarization in vivo is so far not available. In the current study, we aimed to use tumor necrosis factor (TNF)-alpha-induced protein 8-like 2 (TIPE2) to regulate macrophage polarization in vitro and in vivo, and to study the effects on experimental SLE. Methods: We prepared adeno-associated virus carrying TIPE2 (AAV-TIPE2). We induced experimental SLE in mice with an activated lymphocyte-derived DNA (ALD-DNA) method. We examined the effects of TIPE2 overexpression on macrophage polarization in vitro, and in vivo in the SLE model. We also examined the effects of TIPE2 overexpression on the severity of SLE, by serum anti-dsDNA autoantibody, renal pathological changes, and urine protein levels. Results: ALD-DNA induced SLE-like features in mice, manifested by induction of serum anti-dsDNA autoantibody, renal pathological changes, and increases in urine protein levels. TIPE2 overexpression by AAV-TIPE2 induced macrophage polarization to a M2 phenotype, in vitro and in vivo in the SLE mouse model. TIPE2 overexpression significantly decreased SLE severity. Conclusion: TIPE2 alleviates experimental SLE through induction of macrophage polarization to a M2 phenotype, which may be used as a promising therapeutic strategy for treating SLE.

Extracellular, but not intracellular HMGB1, facilitates self-DNA induced macrophage activation via promoting DNA accumulation in endosomes and contributes to the pathogenesis of lupus nephritis

Systemic lupus erythematosus (SLE) is a typical autoimmune disease with unclear etiology. Aberrant self-DNA recognition has been proven critical for the initiation of excessive immune responses in lupus. Consistently, we previously reported that a sort of self-DNA, activated lymphocyte-derived DNA (ALD-DNA) could potentially induce macrophage activation, which contributes to the pathogenesis of murine lupus nephritis. However, the mechanism underlying the self-DNA recognition needs to be further elucidated. Serum level of high mobility group box 1 (HMGB1) has been reported significantly increased both in SLE patients and in lupus mice. Considering its DNA-binding and pro-inflammatory properties, we assumed that HMGB1 might play a role in the processes of ALD-DNA recognition and subsequent activation of macrophages. Here, we found that HMGB1 level was robustly elevated in macrophages following ALD-DNA stimulation via a TLR9 dependent manner. More interestingly, only extracellular but not intracellular HMGB1 could significantly facilitate the ALD-DNA induced macrophage activation, as functional blockade of extracellular HMGB1 efficiently abolished the inflammatory cytokines including IL-6, IL-10, TNF-α, and MCP-1 in ALD-DNA induced macrophages. This effect of extracellular HMGB1 to promote inflammatory cytokine production was associated with its property of accelerating and increasing the accumulation of ALD-DNA in endosomes, which might benefit the DNA recognition by endosome related DNA sensors and promote the subsequent macrophage activation. The pathological role of released HMGB1 was also verified in ALD-DNA induced lupus mice by blocking the serum HMGB1 with glycyrrhizin, which led to the significantly alleviated lupus nephritis as shown decreased inflammatory cytokines, anti-double-stranded DNA (anti-dsDNA) antibodies, urine protein, glomerular IgG and C3 deposition as well as relieved renal histopathology. Taken together, our findings not only better understand in the role of extracellular HMGB1 facilitating ALD-DNA recognition and the subsequent macrophages activation, but also suggest that HMGB1 might serve as an attractive emerging target for SLE treatment.

Macrophage Polarization Modulates Development of Systemic Lupus Erythematosus

Background/Aims: Macrophages have recently been shown to play a critical role in the pathogenesis of systemic lupus erythematosus (SLE). However, the underlying mechanisms remain unclear. Methods: Here, we used an activated lymphocyte-derived DNA (ALD-DNA) method to induce SLE in mice. We used a macrophage-specific eliminator clodronate to selectively deplete macrophages in mice. We isolated macrophages from bone marrow of the mice and used cytokines to differentiate M1 and M2 macrophages, respectively. Adoptive transplantation of M1 or M2 macrophages was performed in clodronate-treated mice. The effects on SLE were evaluated by serum anti-dsDNA autoantibody, by renal pathological changes, and by urine protein levels. Results: ALD-DNA induced SLE-like features in mice, manifested by induction of serum anti-dsDNA autoantibody, by renal pathological changes, and by increases in urine protein levels. Clodronate significantly decreased macrophages in mice, which significantly increased SLE severity. Adoptive transplantation of M2, but not M1 macrophages significantly reduced SLE severity in clodronate- and ALD-DNA-treated mice. Conclusion: M1 and M2 macrophages play different roles in development of SLE. M1 macrophages increase the severity of SLE, while M2 macrophages reduce it. Modulation of macrophage polarity may be an attractive therapy for SLE.

Dynamic expression of microRNAs in M2b polarized macrophages associated with systemic lupus erythematosus.

Macrophage polarization contributes to the initiation and perpetuation of systemic lupus erythematosus (SLE). Our previous study demonstrated that M2b polarized macrophages induced by activated lymphocyte-derived DNA (ALD-DNA) have a crucial function in the initiation and progress of SLE disease. Accumulated data suggest that microRNAs (miRNAs) serve as critical regulators to control macrophage polarization. To investigate miRNA regulation during macrophage M2b polarization of SLE, miRNA microarrays of murine bone marrow derived macrophages (BMDMs) were performed following stimulation with ALD-DNA for 6 and 36 h. Over 11% of the 1111 analyzed miRNAs appeared differentially expressed during ALD-DNA triggered macrophage M2b polarization. Cluster analysis revealed certain patterns in miRNA expression that are closely linked to ALD-DNA induced macrophage M2b polarization. Analysis of the network structure showed that the predicted functions of the differentially regulated miRNAs at 6h are significantly associated with inflammatory response and disease. Differentially regulated miRNAs identified at 36 h were determined to be significantly related to cell proliferation by biological network analysis. In this study, dynamic miRNA expression patterns and network analysis are described for the first time during ALD-DNA induced macrophage M2b polarization. The data not only provide a better understanding of miRNA-mediated macrophage polarization but also demonstrate the future therapeutic potential of targeting miRNAs in SLE patients.

Mannose-Binding Lectin Blunts Macrophage Polarization and Ameliorates Lupus Nephritis

BackgroundDeficiency in clearance of self nuclear antigens, including DNA, is the hallmark of systemic lupus erythematosus (SLE), a chronic autoimmnue disease characterized by the production of various autoantibodies, immune complex deposition and severe organ damage. Our previous studies revealed that administration of syngeneic BALB/c mice with activated lymphocyte-derived DNA (ALD-DNA) could induce SLE disease. Mannose-binding lectin (MBL), a secreted pattern recognition receptor with binding activity to DNA, has been proved to be a modulator of inflammation, but whether MBL takes responsibility for DNA clearance, modulates the DNA-mediated immune responses, and is involved in the development of DNA-induced SLE disease remain poorly understood.Methodology/Principal FindingsThe levels of serum MBL significantly decreased in lupus mice induced by ALD-DNA and were negatively correlated with SLE disease. MBL blunted macrophage M2b polarization by inhibiting the MAPK and NF-κB signaling while enhancing the activation of CREB. Furthermore, MBL suppressed the ability of ALD-DNA–stimulated macrophages to polarize T cells toward Th1 cells and Th17 cells. Importantly, MBL supplement in vivo could ameliorate lupus nephritis.Conclusion/SignificanceThese results suggest MBL supplement could alleviate SLE disease and might imply a potential therapeutic strategy for DNA-induced SLE, which would further our understanding of the protective role of MBL in SLE disease.

Production of anti-double-stranded DNA antibodies in activated lymphocyte derived DNA induced lupus model was dependent on CD4+T cells

Our previous study demonstrated that activated lymphocyte derived DNA (ALD-DNA) could function as an autoantigen to induce production of anti-double-stranded DNA (anti-dsDNA) antibodies in syngeneic BALB/c mice. Here we carefully evaluated the potential role of T cells in the induction of anti-dsDNA antibody. We demonstrated that ALD-DNA could effectively induce production of anti-dsDNA antibodies in vivo and in vitro. In contrast, ALD-DNA could not induce the generation of anti-dsDNA antibodies in nude mice. We further showed that in vivo depletion of CD3(+) T cells blocked the induction of anti-dsDNA antibodies in BALB/c mice. Notably, we demonstrated that CD4(+) but not CD8(+) T cells conferred ALD-DNA to induce anti-dsDNA antibodies. Finally, we demonstrated that adoptive transfer of CD4(+) T cells could rescue ALD-DNA induced anti-dsDNA antibodies in nude mice. Our results suggested that T helper cells were required for ALD-DNA to induce anti-dsDNA antibodies. These findings could further our understanding about the immunogenic properties of DNA and throw new light on SLE pathogenesis.

Amelioration of Lupus Nephritis by Serum Amyloid P Component Gene Therapy with Distinct Mechanisms Varied from Different Stage of the Disease

BackgroundOur previous study revealed that administration of syngeneic female BALB/c mice with excessive self activated lymphocyte-derived DNA (ALD-DNA) could induce systemic lupus erythematosus (SLE) disease, indicating that overload of self-DNA might exceed normal clearance ability and comprise the major source of autoantigens in lupus mice. Serum amyloid P component (SAP), an acute-phase serum protein with binding reactivity to DNA in mice, was proved to promote the clearance of free DNA and prevent mice against self-antigen induced autoimmune response. It is reasonable to hypothesize that SAP treatment might contribute to alleviation of SLE disease, whereas its role in ALD-DNA-induced lupus nephritis is not fully understood.Methodology/Principal FindingsThe ratios of SAP to DNA significantly decreased and were negatively correlated with the titers of anti-dsDNA antibodies in ALD-DNA-induced lupus mice, indicating SAP was relatively insufficient in lupus mice. Herein a pcDNA3-SAP plasmid (pSAP) was genetically constructed and intramuscularly injected into BALB/c mice. It was found that SAP protein purified from the serum of pSAP-treated mice bound efficiently to ALD-DNA and inhibited ALD-DNA-mediated innate immune response in vitro. Treatment of ALD-DNA-induced lupus mice with pSAP in the early stage of SLE disease with the onset of proteinuria reversed lupus nephritis via decreasing anti-dsDNA autoantibody production and immune complex (IC) deposition. Further administration of pSAP in the late stage of SLE disease that had established lupus nephritis alleviated proteinuria and ameliorated lupus nephritis. This therapeutic effect of SAP was not only attributable to the decreased levels of anti-dsDNA autoantibodies, but also associated with the decreased infiltration of lymphocytes and the reduced production of inflammatory markers.Conclusion/SignificanceThese results suggest that SAP administration could effectively alleviated lupus nephritis via modulating anti-dsDNA antibody production and the inflammation followed IC deposition, and SAP-based intervening strategy may provide new approaches for treating SLE disease.

Blockade of Notch1 Signaling Alleviates Murine Lupus via Blunting Macrophage Activation and M2b Polarization

Patients with systemic lupus erythematosus (SLE) are found to be accompanied with innate immunity dysregulation including abnormally macrophage activation. But the functional polarization of the activated macrophages and its underlying molecular mechanism during the pathogenesis of SLE remains unknown. As an important local cellular interaction mechanism responsible for cell fate determination, Notch signaling is reported to exert crucial functions in the development and differentiation of various immunocytes, whereas its role in macrophage polarization is not fully understood. In this study, in the SLE murine model generated by immunization with activated lymphocyte-derived DNA (ALD-DNA), infiltrated macrophages in the nephritic tissues were found to exhibit activation and M2b functional polarization. Notch1 signaling activity was significantly upregulated in the ALD-DNA-induced M2b macrophages in vitro and in vivo. Furthermore, ALD-DNA-induced M2b polarization was found to be dependent on enhanced Notch1 signaling through accelerating NF-kappaB p50 translocation into the nucleus mediated by PI3K and MAPK pathways. Moreover, blockade of Notch1 signaling with gamma-secretase inhibitor treatment before or after the disease initiation could ameliorate murine lupus through impeding macrophage M2b polarization. Our results implied that Notch1 signaling-dependent macrophage M2b polarization might play a pivotal role in the pathogenesis of SLE, which could provide Notch1 signaling blockade as a potential therapeutic approach for SLE disease.

DNA hypomethylation is crucial for apoptotic DNA to induce systemic lupus erythematosus-like autoimmune disease in SLE-non-susceptible mice

Systemic lupus erythematosus (SLE) is characterized by serological presence of anti-double-stranded DNA (dsDNA) antibodies and its pathogenesis remains unclarified. Our previous work found that syngeneic activated lymphocyte-derived DNA (ALD-DNA) induced SLE-like autoimmune disease in the SLE-non-prone BALB/c mice. Here, the biological and chemical characteristics of the somatic DNA were focused upon to investigate their contribution to the autoimmunity induction to provide clues for the understanding of the pathogenesis of SLE in non-susceptible strains. Induction of anti-dsDNA antibodies, glomerulonephritis and proteinuria was evaluated in BALB/c mice after subcutaneous immunization with apoptotic DNA (annexin-V+) extracted from concanavalin A or UV-treated apoptotic splenocytes or necrotic DNA from necrotic splenocytes. The hypomethylated apoptotic DNA and the normal DNA were then methylated and demethylated, respectively, by CpG methylase or 5-azacytidine treatment to re-evaluate their immunogenicity in BALB/c mice. It was apoptotic but not necrotic DNA that induced SLE-like autoimmune disease and the level of apoptotic DNA was associated with the level of anti-dsDNA antibodies. The apoptotic DNA exhibited significantly lower methylation levels than the normal DNA. Methylation of the hypomethylated apoptotic DNA significantly impaired its ability to induce anti-dsDNA antibodies and proteinuria, while demethylation of the normal or necrotic DNA endowed them with the immunogenicity to induce the SLE-like syndrome. Our study provides direct evidence showing that DNA hypomethylation is essential for apoptotic DNA to induce SLE-like autoimmune disease in non-susceptible mice, which may help in elucidating the pathogenesis of SLE.