Int J Clin Exp Med 2015;8(10):17430-17440 www.ijcem.com /ISSN:1940-5901/IJCEM0014042
Original Article A20 overexpression alleviates pristine-induced lupus nephritis by inhibiting the NF-κB and NLRP3 inflammasome activation in macrophages of mice Min Li1, Xiaowei Shi1, Tian Qian1, Jian Li1, Zhiqiang Tian2, Bing Ni2, Fei Hao1 1 2
Department of Dermatology, Southwest Hospital, Third Military Medical University, Chongqing, 400038, China; Institute of Immunology PLA, Third Military Medical University, Chongqing 400038, China
Received August 5, 2015; Accepted October 13, 2015; Epub October 15, 2015; Published October 30, 2015 Abstract: Background: Lupus nephritis is an autoimmune inflammatory disease and urgently needs effective antiinflammation therapies. A20, tumor necrosis factor alpha induced protein 3 (TNFAIP3), is a key negative regulator of inflammation, however whether A20 can regulate lupus nephritis has not been clarified. This study aimed at investigating the potential therapeutic effect of A20 on renal inflammation in mouse pristine model oflupus. Methodology/ Principal Findings: Female BALB/c mice were intraperitoneally injected with pristine to establish lupus renal injury. The levels of serum IL-1β, IL-6 and autoantibodies and the degrees of renal injury and CCL2 and F4/80 levels were measured. The levels of the NF-κB and NLRP3 inflammasome activation in peritoneal macrophages were determined. We found that injection with pristine increased the levels of serum IL-1β, IL-6, autoantibodies and CCL20 and F4/80 expression in the kidney and induced renal injury, accompanied by enhancing the NF-κB and NLRP3 inflammasome activation in macrophages of mice. In contrast, treatment with Ad-A20, but not with Ad-control, significantly mitigated pristine-induced inflammatory responses and renal injury,and reduced the NF-κB and NLRP3 inflammasome activation in macrophages in mice. Conclusion/Significance: Our data indicated that induction of A20 overexpression inhibited pristane induced lupus inflammation and renal injury in mice and may be a new therapeutic strategy for treatment of lupus nephritis. Keywords: A20, pristine, cytokine, lupus nephritis, adenovirus, macrophages, NF-κB, NLRP3 inflammasome, mice
Introduction Systemic lupus erythematosus (SLE) is an autoimmune disease predominantly affecting females. During the process of SLE, loss of tolerance to nucleic acids and their interacting proteins results in the production of pathogenic autoantibodies that cause inflammation and tissue damage [1]. Although advances in the medical management of infections and renal failure have improved the survival of patients with lupus, the efficacy of available therapeutic strategies for lupus nephritis and SLE is limited. The current broad-spectrum immunosuppressive agents are not always adequate to control clinical symptoms or prevent disease flares. Chronic inflammation in SLE patients can induce aberrant activation of signaling cascades that confer resistance to immunosuppressive drugs and deteriorate tissue damage
[2, 3]. Therefore, development of new therapies for control of systemic inflammation is of significance in management of patients with SLE. Macrophages act as professional antigen presenting cells and are crucial for the development of SLE and lupus nephritis [4]. Macrophages can infiltrate in the kidney and secrete pro-inflammatory cytokines, such as IL-1β and IL-6, and chemokines, such as CCL2, which are important for the development and progression of nephritis [5, 6]. Furthermore, inflammatory stimuli can activate the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) expression, which interacts with apoptosis-associated speck-like protein containing a CARD (ASC) to recruit caspase-1, leading to inflammasome activation [7]. In addition, the enhanced NF-κB signaling and subsequent activation of the NLRP3 promote inflammatory
A20 overexpression alleviates pristine-induced lupus nephritis cytokine production in macrophages [8]. Hence, down-regulation of NLRP3 inflammasome activation and the NF-κB signaling may be critical for control of macrophage-related inflammatory.
plasmid was co-transfected with packaging helper plasmids (Oobio) into 293T cells to generate the recombinant Ad-A20 and control Ad-control. The generated recombinant Ad-A20 and control Ad-control were titered.
A20, also known as tumor necrosis factor alpha induced protein 3 (TNFAIP3) is an anti-inflammatory factor induced by TNF [9]. A20 can inhibit TNFα-induced cell apoptosis and the NF-κB and IRF activation [10]. Mice deficient for A20 expression spontaneously develop severe inflammation and cachexia, are hypersensitive to both lipopolysaccharide and TNF, and die prematurely [11]. Previous studies have shown that A20 inhibits the pathogenic process of inflammatory diseases, such as myocarditis [12], hepatitis [13], and arthritis [14]. However, the effect of A20 on inflammation during the process of lupus nephritis has not been clarified.
Generation and treatment of mouse model of SLE
In this study, we generated recombinant adenovirus to induce A20 overexpression and examined the therapeutic effect of A20 overexpression on systemic inflammation, autoantibody responses, renal inflammation and injury in mouse pristine model of lupus nephritis. Furthermore, we explored the potential mechanisms underlying the action of A20 in regulating the NF-κB and NLRP3 inflammasome activation in peritoneal macrophages of mice. Materials and methods Mice Female BALB/c mice at 4 weeks of age were purchased from the Experimental Animal Centre of the Third Military Medical Universityand housed in a specific pathogen-free facility at 25±2°C and 40-70% relative humidity with a 12-h day/night lighting cycle and free access to standard rodent chew and filtered water. After being acclimated for 2 weeks, the mice were used for experiment. The experimental protocol was proved by the Ethics Committee of the Third Military Medical University. Preparation of recombinant adenoviruses TNFAIP3 cDNA was from Oobio (Shanghai, China) and cloned into the plasmid of pADVMCMV-3FLAG-IRES-EGFP (AddGene, US). After being sequenced, the recombinant or control
17431
Individual BALB/c mice at 6-8 weeks of age were injected intraperitoneally (i.p) with 0.5 ml pristine (Sigma-Aldrich) or PBS. Three months later, the pristine-injected mice were randomized and injected i.p with 1.0×109 plaque forming units (PFU) of Ad-A20, Ad-control or PBS (100 μl, n=6-8 per group). The PBS-injected mice served as the healthy PBS control group. One month after treatment with Adenovirus, 24-h urinary and blood samples from individual mice were collected for measurement of urinary albumin and preparation of serum sample, respectively. The mice were sacrificed and their peritoneal macrophages were harvested by peritoneal lavage. Furthermore, their kidney tissues were immediately frozen in liquate nitrogen and stored in -80°C or fixed with 10% formalin overnight and paraffin-embedded, respectively. Preparation of murine peritoneal macrophages Peritoneal macrophages from healthy and pristine-injected mice were harvested by peritoneal lavage with cold DMEM medium supplemented with 1% penicillin/streptomycin. The cells were washed, and re-suspended in DMEM medium supplemented with 10% FBS (complete medium). Peritoneal macrophages were incubated in complete medium at 37°C 5% CO2 for 2 h, and after removing non-adherent cells, the adherent cells were cultured in freshDMEM medium as macrophages. Enzyme-linked immunosorbent assay (ELISA) The levels of serum IL-1β and IL-6 in individual mice were measured using mouse cytokine kits (Invitrogen/BioSource International, Camarillo, USA). Similarly, the levels of serum IgG, antinRNP, and anti-dsDNA antibodies were also measured using specific ELISA kits, according to the manufacturer’s instructions (Alpha Diagnostic, San Antonio, TX). Briefly, serum samples (50 μL each) at 1:4 dilutions were added in triplicate into 96-well plates that had
Int J Clin Exp Med 2015;8(10):17430-17440
A20 overexpression alleviates pristine-induced lupus nephritis been coated with specific antibodies and incubated at room temperature for 2 h, and after being washed, the bound antigens were detected with biotinylated antibodies at room temperature for 0.5 h. Subsequently, the bound biotinylated antibodies were detected with horseradish peroxidase (HRP)-conjugated Streptavidin and chromogen reagent, followed by reading the absorbance at 450 nm in an ELISA reader. The levels of urinary albumin in individual 24-h urinal samples were analyzed using a mouse albumin ELISA quantification kit (Bethyl Laboratories, USA).
anti-p-IκB, anti-IκB, anti-F4/80, anti-CCL2 (Abcam, Cambridge, USA), anti-Caspase-1p20, anti-p-NF-κBp65, anti-NF-κBp65, anti-GAPDH and anti-fibrillarin (Santa Cruz Biotechnology, Santa Cruz, USA) at 4°C overnight. After being washed, the bound antibodies were detected with HRP-conjugated secondary antibodies (Santa Cruz Biotechnology) for 1 h and visualized using the enhanced chemiluminescence detection system (Millipore, Minnesota, USA). The relative levels of target protein expression to the internal control were qualified using the Image J analysis software.
Histological examination of the kidney injury
Statistical analysis
Paraffin-embedded renal tissue sections (4 μm) were stained with hematoxylin and eosin (H&E) and examined under a light microscope. The pathologic changes in the kidney tissues were evaluated for the glomerular and tubulointerstitial activity scores of 8 fields of each mouse in a blinded manner using a scale of 0-3, as reported previously [15].
Quantitative data are expressed as mean ± SD. The difference among groups was analyzed by the Mann-Whitney u test or one-way ANOVA when the data were normally distributed using the GraphPad Prism (Version 5.0). A p value of
Original Article A20 overexpression alleviates pristine-induced lupus nephritis by inhibiting the NF-κB and NLRP3 inflammasome activation in macrophages of mice Min Li1, Xiaowei Shi1, Tian Qian1, Jian Li1, Zhiqiang Tian2, Bing Ni2, Fei Hao1 1 2
Department of Dermatology, Southwest Hospital, Third Military Medical University, Chongqing, 400038, China; Institute of Immunology PLA, Third Military Medical University, Chongqing 400038, China
Received August 5, 2015; Accepted October 13, 2015; Epub October 15, 2015; Published October 30, 2015 Abstract: Background: Lupus nephritis is an autoimmune inflammatory disease and urgently needs effective antiinflammation therapies. A20, tumor necrosis factor alpha induced protein 3 (TNFAIP3), is a key negative regulator of inflammation, however whether A20 can regulate lupus nephritis has not been clarified. This study aimed at investigating the potential therapeutic effect of A20 on renal inflammation in mouse pristine model oflupus. Methodology/ Principal Findings: Female BALB/c mice were intraperitoneally injected with pristine to establish lupus renal injury. The levels of serum IL-1β, IL-6 and autoantibodies and the degrees of renal injury and CCL2 and F4/80 levels were measured. The levels of the NF-κB and NLRP3 inflammasome activation in peritoneal macrophages were determined. We found that injection with pristine increased the levels of serum IL-1β, IL-6, autoantibodies and CCL20 and F4/80 expression in the kidney and induced renal injury, accompanied by enhancing the NF-κB and NLRP3 inflammasome activation in macrophages of mice. In contrast, treatment with Ad-A20, but not with Ad-control, significantly mitigated pristine-induced inflammatory responses and renal injury,and reduced the NF-κB and NLRP3 inflammasome activation in macrophages in mice. Conclusion/Significance: Our data indicated that induction of A20 overexpression inhibited pristane induced lupus inflammation and renal injury in mice and may be a new therapeutic strategy for treatment of lupus nephritis. Keywords: A20, pristine, cytokine, lupus nephritis, adenovirus, macrophages, NF-κB, NLRP3 inflammasome, mice
Introduction Systemic lupus erythematosus (SLE) is an autoimmune disease predominantly affecting females. During the process of SLE, loss of tolerance to nucleic acids and their interacting proteins results in the production of pathogenic autoantibodies that cause inflammation and tissue damage [1]. Although advances in the medical management of infections and renal failure have improved the survival of patients with lupus, the efficacy of available therapeutic strategies for lupus nephritis and SLE is limited. The current broad-spectrum immunosuppressive agents are not always adequate to control clinical symptoms or prevent disease flares. Chronic inflammation in SLE patients can induce aberrant activation of signaling cascades that confer resistance to immunosuppressive drugs and deteriorate tissue damage
[2, 3]. Therefore, development of new therapies for control of systemic inflammation is of significance in management of patients with SLE. Macrophages act as professional antigen presenting cells and are crucial for the development of SLE and lupus nephritis [4]. Macrophages can infiltrate in the kidney and secrete pro-inflammatory cytokines, such as IL-1β and IL-6, and chemokines, such as CCL2, which are important for the development and progression of nephritis [5, 6]. Furthermore, inflammatory stimuli can activate the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) expression, which interacts with apoptosis-associated speck-like protein containing a CARD (ASC) to recruit caspase-1, leading to inflammasome activation [7]. In addition, the enhanced NF-κB signaling and subsequent activation of the NLRP3 promote inflammatory
A20 overexpression alleviates pristine-induced lupus nephritis cytokine production in macrophages [8]. Hence, down-regulation of NLRP3 inflammasome activation and the NF-κB signaling may be critical for control of macrophage-related inflammatory.
plasmid was co-transfected with packaging helper plasmids (Oobio) into 293T cells to generate the recombinant Ad-A20 and control Ad-control. The generated recombinant Ad-A20 and control Ad-control were titered.
A20, also known as tumor necrosis factor alpha induced protein 3 (TNFAIP3) is an anti-inflammatory factor induced by TNF [9]. A20 can inhibit TNFα-induced cell apoptosis and the NF-κB and IRF activation [10]. Mice deficient for A20 expression spontaneously develop severe inflammation and cachexia, are hypersensitive to both lipopolysaccharide and TNF, and die prematurely [11]. Previous studies have shown that A20 inhibits the pathogenic process of inflammatory diseases, such as myocarditis [12], hepatitis [13], and arthritis [14]. However, the effect of A20 on inflammation during the process of lupus nephritis has not been clarified.
Generation and treatment of mouse model of SLE
In this study, we generated recombinant adenovirus to induce A20 overexpression and examined the therapeutic effect of A20 overexpression on systemic inflammation, autoantibody responses, renal inflammation and injury in mouse pristine model of lupus nephritis. Furthermore, we explored the potential mechanisms underlying the action of A20 in regulating the NF-κB and NLRP3 inflammasome activation in peritoneal macrophages of mice. Materials and methods Mice Female BALB/c mice at 4 weeks of age were purchased from the Experimental Animal Centre of the Third Military Medical Universityand housed in a specific pathogen-free facility at 25±2°C and 40-70% relative humidity with a 12-h day/night lighting cycle and free access to standard rodent chew and filtered water. After being acclimated for 2 weeks, the mice were used for experiment. The experimental protocol was proved by the Ethics Committee of the Third Military Medical University. Preparation of recombinant adenoviruses TNFAIP3 cDNA was from Oobio (Shanghai, China) and cloned into the plasmid of pADVMCMV-3FLAG-IRES-EGFP (AddGene, US). After being sequenced, the recombinant or control
17431
Individual BALB/c mice at 6-8 weeks of age were injected intraperitoneally (i.p) with 0.5 ml pristine (Sigma-Aldrich) or PBS. Three months later, the pristine-injected mice were randomized and injected i.p with 1.0×109 plaque forming units (PFU) of Ad-A20, Ad-control or PBS (100 μl, n=6-8 per group). The PBS-injected mice served as the healthy PBS control group. One month after treatment with Adenovirus, 24-h urinary and blood samples from individual mice were collected for measurement of urinary albumin and preparation of serum sample, respectively. The mice were sacrificed and their peritoneal macrophages were harvested by peritoneal lavage. Furthermore, their kidney tissues were immediately frozen in liquate nitrogen and stored in -80°C or fixed with 10% formalin overnight and paraffin-embedded, respectively. Preparation of murine peritoneal macrophages Peritoneal macrophages from healthy and pristine-injected mice were harvested by peritoneal lavage with cold DMEM medium supplemented with 1% penicillin/streptomycin. The cells were washed, and re-suspended in DMEM medium supplemented with 10% FBS (complete medium). Peritoneal macrophages were incubated in complete medium at 37°C 5% CO2 for 2 h, and after removing non-adherent cells, the adherent cells were cultured in freshDMEM medium as macrophages. Enzyme-linked immunosorbent assay (ELISA) The levels of serum IL-1β and IL-6 in individual mice were measured using mouse cytokine kits (Invitrogen/BioSource International, Camarillo, USA). Similarly, the levels of serum IgG, antinRNP, and anti-dsDNA antibodies were also measured using specific ELISA kits, according to the manufacturer’s instructions (Alpha Diagnostic, San Antonio, TX). Briefly, serum samples (50 μL each) at 1:4 dilutions were added in triplicate into 96-well plates that had
Int J Clin Exp Med 2015;8(10):17430-17440
A20 overexpression alleviates pristine-induced lupus nephritis been coated with specific antibodies and incubated at room temperature for 2 h, and after being washed, the bound antigens were detected with biotinylated antibodies at room temperature for 0.5 h. Subsequently, the bound biotinylated antibodies were detected with horseradish peroxidase (HRP)-conjugated Streptavidin and chromogen reagent, followed by reading the absorbance at 450 nm in an ELISA reader. The levels of urinary albumin in individual 24-h urinal samples were analyzed using a mouse albumin ELISA quantification kit (Bethyl Laboratories, USA).
anti-p-IκB, anti-IκB, anti-F4/80, anti-CCL2 (Abcam, Cambridge, USA), anti-Caspase-1p20, anti-p-NF-κBp65, anti-NF-κBp65, anti-GAPDH and anti-fibrillarin (Santa Cruz Biotechnology, Santa Cruz, USA) at 4°C overnight. After being washed, the bound antibodies were detected with HRP-conjugated secondary antibodies (Santa Cruz Biotechnology) for 1 h and visualized using the enhanced chemiluminescence detection system (Millipore, Minnesota, USA). The relative levels of target protein expression to the internal control were qualified using the Image J analysis software.
Histological examination of the kidney injury
Statistical analysis
Paraffin-embedded renal tissue sections (4 μm) were stained with hematoxylin and eosin (H&E) and examined under a light microscope. The pathologic changes in the kidney tissues were evaluated for the glomerular and tubulointerstitial activity scores of 8 fields of each mouse in a blinded manner using a scale of 0-3, as reported previously [15].
Quantitative data are expressed as mean ± SD. The difference among groups was analyzed by the Mann-Whitney u test or one-way ANOVA when the data were normally distributed using the GraphPad Prism (Version 5.0). A p value of
