Omega-3 Docosahexaenoic Acid (DHA) Impedes Silica-Induced Macrophage Corpse Accumulation by Attenuating Cell Death and Potentiating Efferocytosis.

Airway exposure of lupus-prone NZBWF1 mice to crystalline silica (cSiO2), a known trigger of human autoimmune disease, elicits sterile inflammation and alveolar macrophage death in the lung that, in turn, induces early autoimmune onset and accelerates lupus progression to fatal glomerulonephritis. Dietary supplementation with docosahexaenoic acid (DHA), a marine ω-3 polyunsaturated fatty acid (PUFA), markedly ameliorates cSiO2-triggered pulmonary, systemic, and renal manifestations of lupus. Here, we tested the hypothesis that DHA influences both cSiO2-induced death and efferocytotic clearance of resultant cell corpses using three murine macrophage models: (i) primary alveolar macrophages (AM) isolated from NZBWF1 mice; (ii) self-renewing AM-like Max Planck Institute (MPI) cells isolated from fetuses of C57BL/6 mice, and (iii) RAW 264.7 murine macrophages, a virus-transformed cell line derived from BALB/c mice stably transfected with the inflammasome adaptor protein ASC (RAW-ASC). Incubation with cSiO2 at 25 and 50 µg/ml for 6 h was found to dose-dependently induce cell death (p < 0.05) in all three models as determined by both acridine orange/propidium iodide staining and release of lactate dehydrogenase into cell culture supernatant. Pre-incubation with DHA at a physiologically relevant concentration (25 µM) significantly reduced cSiO2-induced death (p < 0.05) in all three models. Cell death induction by cSiO2 alone and its suppression by DHA were primarily associated with caspase-3/7 activation, suggestive of apoptosis, in AM, MPI, and RAW-ASC cells. Fluorescence microscopy revealed that all three macrophage models were similarly capable of efferocytosing RAW-ASC target cell corpses. Furthermore, MPI effector cells could likewise engulf RAW-ASC target cell corpses elicited by treatment with staurosporine (apoptosis), LPS, and nigericin (pyroptosis), or cSiO2. Pre-incubation of RAW-ASC target cells with 25 µM DHA prior to death induced by these agents significantly enhanced their efferocytosis (p < 0.05) by MPI effector cells. In contrast, pre-incubating MPI effector cells with DHA did not affect engulfment of RAW-ASC target cells pre-incubated with vehicle. Taken together, these findings indicate that DHA at a physiologically relevant concentration was capable of attenuating macrophage death and could potentiate efferocytosis, with the net effect of reducing accumulation of cell corpses capable of eliciting autoimmunity.

Requisite Omega-3 HUFA Biomarker Thresholds for Preventing Murine Lupus Flaring.

Lupus is a systemic autoimmune disease typified by uncontrolled inflammation, disruption of immune tolerance, and intermittent flaring - events triggerable by environmental factors. Preclinical and clinical studies reveal that consumption of the marine ω-3 highly unsaturated fatty acids (HUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) might be used as a precision nutrition intervention to lessen lupus symptoms. The anti-inflammatory and pro-resolving effects of ω-3 HUFAs are inextricably linked to their presence in membrane phospholipids. The ω-3 HUFA score, calculated as [100 × (ω-3 HUFAs/(ω-3 HUFAs + ω-6 HUFAs))] in red blood cells (RBCs), and the Omega-3 Index (O3I), calculated as [100 × ((DHA+EPA)/total fatty acids)] in RBCs, are two biomarkers potentially amenable to relating tissue HUFA balance to clinical outcomes in individuals with lupus. Using data from three prior preclinical DHA supplementation studies, we tested the hypothesis that the ω-3 HUFA score and the O3I inversely correlate with indicators of autoimmune pathogenesis in the cSiO2-triggered lupus flaring model. The three studies employed both low and high fat rodent diets, as well as more complex diets emulating the U.S. dietary pattern. The ω-3 HUFA scores in RBCs were comparatively more robust than the O3I at predicting HUFA balances in the kidney, liver, spleen, and lung. Importantly, increases in both the ω-3 HUFA score (>40%) and the O3I (>10%) were strongly associated with suppression of cSiO2-triggered (1) expression of interferon-regulated genes, proinflammatory cytokine production, leukocyte infiltration, and ectopic lymphoid structure development in the lung, (2) pulmonary and systemic autoantibody production, and (3) glomerulonephritis. Collectively, these findings identify achievable ω-3 HUFA scores and O3I thresholds that could be targeted in future human intervention studies querying how ω-3 HUFA consumption influences lupus and other autoimmune diseases.

Accelerated and Severe Lupus Nephritis Benefits From M1, an Active Metabolite of Ginsenoside, by Regulating NLRP3 Inflammasome and T Cell Functions in Mice.

Chinese herbal medicines used in combination have long-term been shown to be mild remedies with "integrated effects." However, our study provides the first demonstration that M1, an active metabolite of ginsenoside, exerted its dramatic therapeutic effects on accelerated and severe lupus nephritis (ASLN) mice, featuring acute renal function impairment, heavy proteinuria, high serum levels of anti-dsDNA, and high-grade, diffuse proliferative renal lesions. In the present study, NZB/WF1 mice were given injections of lipopolysaccharide to induce the ASLN model. M1 (30 mg/kg) was then administered to the mice by gavage daily, and the mice were sacrificed on week 3 and week 5 after the induction of disease. To identify the potential mechanism of action for the pure compound, levels of NLRP3 inflammasome activation in bone marrow-derived dendritic cells (BMDCs), podocytes and macrophages, and antigen-specific T cell activation in BMDCs were determined in addition to mechanistic experiments in vivo. Treatment with M1 dramatically improved renal function, albuminuria and renal lesions and reduced serum levels of anti-dsDNA in the ASLN mice. These beneficial effects with M1 treatment involved the following cellular and molecular mechanistic events: [1] inhibition of NLRP3 inflammasome associated with autophagy induction, [2] modulation of T help cell activation, and [3] induction of regulatory T cell differentiation. M1 improved the ASLN mice by blunting NLRP3 inflammasome activation and differentially regulating T cell functions, and the results support M1 as a new therapeutic candidate for LN patients with a status of abrupt transformation of lower-grade (mesangial) to higher-grade (diffuse proliferative) nephritis.

The Root Extract of Gentiana macrophylla Pall. Alleviates B19-NS1-Exacerbated Liver Injuries in NZB/W F1 Mice.

The nonstructural protein NS1 of human parvovirus B19 (B19) is known to exacerbate disease activity in systemic lupus erythematosus (SLE). However, no specific medicine for B19 infection is available. The roots of Gentiana macrophylla Pall. (GM), the traditional Chinese medicine "Qinjiao," have been used for centuries to treat rheumatic disease, including SLE. Herein, we aimed to investigate the effects of GM root extract (100 and 300 mg/kg body weight) on B19-NS1-exacerbated liver injury in NZB/W F1 mice; liver tissues were assessed by hematoxylin-eosin staining and immunoblotting. The GM root extract significantly decreased B19-NS1-exacerbated liver inflammation by suppressing the expressions of hepatic inducible nitric oxide synthase, cyclooxygenase type 2 (COX-2), interleukin (IL)-1ß proteins, values of serum asparate transaminase (AST) and alanine transaminase (ALT), and lymphocyte infiltration (P < .05). It also significantly reduced the B19-NS1-exacerbated hepatic matrix metalloproteinase-9 (MMP-9) and urokinase plasminogen activator (uPA) expressions by downregulating tumor necrosis factor (TNF)-α/NF-κB (p65) signaling. These findings suggest a therapeutic potential of GM root extract against B19-NS1-exacerbated liver inflammation in SLE.

Lactoferrin Increases Antioxidant Activities and Ameliorates Hepatic Fibrosis in Lupus-Prone Mice Fed with a High-Cholesterol Diet.

Lactoferrin (LF) has beneficial effects against various diseases. However, the effects of LF on liver fibrosis in systematic lupus erythematosus (SLE) are unknown. In this study, NZB/W F1 mice were utilized to investigate the effects of LF on SLE. Experiments reveal that LF significantly increases glutathione and 1,1-diphenyl-2-picryl-hydrazyl levels and significantly decreased malondialdehyde levels in both serum and liver in NZB/W F1 mice. LF also lowered matrix metalloproteinase-9 activity and liver inflammatory indices, such as aminotransferase and alanine aminotransferase. Notably, significantly decreased expression of fibrotic related molecules, including transforming growth factor (TGF)-ß1, tumor necrosis factor-α, interleukin-1ß, and TGF-ß1 receptor, were observed in the livers of NZB/W F1 mice that had been treated with LF. Significantly, suppressed Smad2/3 signaling, α-smooth muscle actin, and collagen deposition were also detected. These findings reveal that LF has beneficial effects on SLE by increasing antioxidant activities and ameliorating liver inflammation and fibrosis, suggesting the therapeutic effectiveness of LF against SLE.

Citral alleviates an accelerated and severe lupus nephritis model by inhibiting the activation signal of NLRP3 inflammasome and enhancing Nrf2 activation.

INTRODUCTION: Lupus nephritis (LN) is a major complication of systemic lupus erythematosus. NLRP3 inflammasome activation, reactive oxygen species (ROS) and mononuclear leukocyte infiltration in the kidney have been shown to provoke the acceleration and deterioration of LN, such as accelerated and severe LN (ASLN). Development of a novel therapeutic remedy based on these molecular events to prevent the progression of the disease is clinically warranted. METHODS: Citral (3,7-dimethyl-2,6-octadienal), a major active compound in a Chinese herbal medicine Litsea cubeba, was used to test its renoprotective effects in a lipopolysaccharide (LPS)-induced mouse ASLN model by examining NLRP3 inflammasome activation, ROS and COX-2 production as well as Nrf2 activation. The analysis of mechanisms of action of Citral also involved its effects on IL-1ß secretion and signaling pathways of NLRP3 inflammasome in LPS-primed peritoneal macrophages or J774A macrophages. RESULTS: Attenuated proteinuria, renal function impairment, and renal histopathology, the latter including intrinsic cell proliferation, cellular crescents, neutrophil influx, fibrinoid necrosis in the glomerulus, and peri-glomerular infiltration of mononuclear leukocytes as well as glomerulonephritis activity score were observed in Citral-treated ASLN mice. In addition, Citral inhibited NLRP3 inflammasome activation and levels of ROS, NAD(P)H oxidase subunit p47(phox), or COX-2, and it enhanced the activation of nuclear factor E2-related factor 2 (Nrf2). In LPS-primed macrophages, Citral reduced ATP-induced IL-1ß secretion and caspase-1 activation, but did not affect LPS-induced NLRP3 protein expression. CONCLUSION: Our data show that Citral alleviates the mouse ASLN model by inhibition of the activation signal, but not the priming signal, of NLRP3 inflammasome and enhanced activation of Nrf2 antioxidant signaling.

The Root Extract of Gentiana macrophylla Pall. Alleviates Cardiac Apoptosis in Lupus Prone Mice.

The roots of the perennial herb Gentiana macrophylla Pall. (GM) are known as Qinjiao, which has been used for centuries to treat systemic lupus erythematosus (SLE). However, little is known about the effects of GM on cholesterol-aggravated cardiac abnormalities in SLE, and the mechanisms thereof. This study investigates whether GM exhibits anti-apoptotic effects, focusing on the left ventricle (LV) of NZB/W F1 mice fed with high-cholesterol diet. The morphology and apoptotic status of ventricular tissues were determined by microscopy and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Levels of apoptotic biomarkers were determined by immunoblotting. The results thus obtained revealed that GM significantly reduced the cholesterol-aggravated apoptosis of LV in NZB/W F1 mice by suppressing both intrinsic and extrinsic apoptotic pathways. Additionally, GM significantly increased the cardiac insulin-like growth factors (IGF)-1 survival signaling and anti-apoptotic proteins in LV tissues. Accordingly, GM is considered to be beneficial in alleviating cholesterol-aggravated cardiac damage in SLE, and therefore constitute an alternative treatment for SLE patients with cardiac abnormalities.

Dual B cell immunotherapy is superior to individual anti-CD20 depletion or BAFF blockade in murine models of spontaneous or accelerated lupus.

OBJECTIVE: To determine whether a combination of B cell depletion and BAFF blockade is more effective than monotherapy in treating models of spontaneous or accelerated systemic lupus erythematosus (SLE) in (NZB × NZW)F1 mice. METHODS: Clinical parameters such as disease progression-free survival, proteinuria, and renal injury were assessed in models of spontaneous, interferon-α (IFNα)-accelerated, or pristane-accelerated lupus in (NZB × NZW)F1 mice. Treatment arms included anti-CD20 (B cell depletion), B lymphocyte stimulator receptor 3 fusion protein (BR-3-Fc) (BAFF blockade), the combination of anti-CD20 and BR-3-Fc, isotype control, or cyclophosphamide. In models of spontaneous, IFNα-accelerated, or pristane-accelerated lupus, mice were treated for 24 weeks, 8 weeks, or 12 weeks, respectively. Peripheral and resident B cell subsets and various autoantibodies were examined. RESULTS: Compared to B cell depletion or BAFF blockade alone, combined therapy significantly improved disease manifestations in all 3 lupus models. In addition, marginal zone B cells, plasmablasts, and circulating and tissue plasma cells were decreased more effectively. Dual B cell immunotherapy also reduced multiple classes of pathogenic autoantibodies, consistent with its observed effectiveness in reducing immune complex-mediated renal injury. CONCLUSION: Dual immunotherapy via B cell depletion and BAFF blockade is more efficacious than single agent immunotherapy in murine SLE models, and this combination treatment is predicted to be an effective strategy for immunotherapy in human SLE.

CCR1 inhibition ameliorates the progression of lupus nephritis in NZB/W mice.

Systemic lupus erythematosus is a chronic inflammatory autoimmune disease, the development of which is characterized by a progressive loss of renal function. Such dysfunction is associated with leukocyte infiltration in the glomerular and tubulointerstitial compartments in both human and experimental lupus nephritis. In this study, we investigated the role of the Ccr1 chemokine receptor in this infiltration process during the progression of nephritis in the lupus-prone New Zealand Black/New Zealand White (NZB/W) mouse model. We found that peripheral T cells, mononuclear phagocytes, and neutrophils, but not B cells, from nephritic NZB/W mice were more responsive to Ccr1 ligands than the leukocytes from younger prenephritic NZB/W mice. Short-term treatment of nephritic NZB/W mice with the orally available Ccr1 antagonist BL5923 decreased renal infiltration by T cells and macrophages. Longer Ccr1 blockade decreased kidney accumulation of effector/memory CD4(+) T cells, Ly6C(+) monocytes, and both M1 and M2 macrophages; reduced tubulointerstitial and glomerular injuries; delayed fatal proteinuria; and prolonged animal lifespan. In contrast, renal humoral immunity was unaffected in BL5923-treated mice, which reflected the unchanged numbers of infiltrated B cells in the kidneys. Altogether, these findings define a pivotal role for Ccr1 in the recruitment of T and mononuclear phagocyte cells to inflamed kidneys of NZB/W mice, which in turn contribute to the progression of renal injury.

Curcumin attenuates lupus nephritis upon interaction with regulatory T cells in New Zealand Black/White mice.

Curcumin has been used in Asian traditional medicine for its medicinal properties. Recent studies have demonstrated that curcumin has antioxidant, anti-tumour and anti-inflammatory activities. The aim of the present study is to investigate the effects of curcumin on established lupus nephritis (LN) in New Zealand Black/White (NZB/W) F1 female mice, in particular, its interaction with regulatory T (Treg) cells. Starting at 18 weeks of age, mice were fed a standard diet or a diet containing 1 % curcumin until the end of the study. The proteinuria level and the serum levels of IgG1, IgG2a and anti-double-stranded DNA (dsDNA) IgG antibodies were measured. Additionally, IgG immune complex deposition in the glomeruli and renal inflammation were compared between curcumin-treated mice and control mice. Curcumin decreased the proteinuria level and serum levels of IgG1, IgG2a and anti-dsDNA IgG antibodies in NZB/W F1 female mice. IgG immune complex deposition in the glomeruli was reduced in curcumin-treated mice. Furthermore, renal inflammation was also decreased after curcumin treatment. Interestingly, these therapeutic effects of curcumin disappeared after Treg depletion by anti-CD25 antibody injection. Curcumin exerted a protective effect against LN in NZB/W F1 mice. We speculate that the protective effects of curcumin in LN may involve, at least in part, its interaction with Treg cells.

Attenuation of nephritis in lupus-prone mice by thalidomide.

OBJECTIVES: Thalidomide has various effects, such as immune modulation, anti-angiogenicity, anti-inflammation and anti-proliferation. Moreover, thalidomide modulates the activity of NF-κB, which can up-regulate the expression of downstream genes involved in the pathophysiology of LN. Here we investigated the efficacy of thalidomide monotherapy or thalidomide plus prednisolone (PL) on nephritis in NZB/WF1 mice at different doses and compared both with a combination therapy of MMF plus PL. METHODS: Forty-three female NZB/WF1 mice were divided into eight groups (untreated; 1.7, 5 or 10 mg/kg of thalidomide alone; 1.7, 5 or 10 mg/kg of thalidomide plus 1.5 mg/kg of PL and 33.3 mg/kg of MMF plus PL). Proteinuria and histological damage were evaluated. Immune complex deposition and nuclear translocation of NF-κB in kidney tissues were assessed by immunofluorescence staining. Serum concentrations of anti-dsDNA and IgG subclasses were also measured. RESULTS: In comparison with untreated mice, mice treated with 10 mg/kg of thalidomide monotherapy showed a significant decrease in proteinuria and significantly lower glomerular and tubular damage scores, comparable to 5 or 10 mg/kg of thalidomide plus PL or MMF plus PL. Also, treatment with 10 mg/kg of thalidomide significantly decreased immune complex accumulation, reduced the serum concentration of anti-dsDNA, IgG2a and IgG2b and inhibited nuclear translocation of NF-κB in kidney tissues, comparable to standard therapy for LN. CONCLUSION: These data suggest that thalidomide might play an anti-inflammatory role in the pathophysiology of LN, and it could serve as a complementary therapy to standard induction regimens for refractory LN.

Genetic variation at a Yin-Yang 1 response site regulates the transcription of cyclin-dependent kinase inhibitor p18INK4C transcript in lupus-prone mice.

We have previously shown that a novel -74 C-to-T mutation in the promoter of the cyclin-dependent kinase inhibitor p18(Ink4c) (p18) gene was associated with a reduced p18 expression in B cells from mice carrying the Sle2c1 lupus susceptibility locus. To determine the function of the -74 C/T single nucleotide polymorphism, we have characterized the proximal promoter of the mouse p18 gene. Functional analysis of the 5' flanking region by sequential deletions revealed crucial elements between -300 and +1, confirming the in silico prediction that the -74 T allele created a novel Yin-Yang 1 (YY-1) binding site adjacent to an existing one common to both alleles. Moreover, we found that YY-1, E2F1, and Sp-1 can synergistically enhance the activity of the p18 promoter. Mutational inactivation revealed that YY-1 binding regulates the p18 activity in an allele-dependent fashion. EMSAs with splenic B cell extracts directly demonstrated that YY-1 binds to the p18 promoter with differences between the C and the T alleles. We also determined in vivo by chromatin immunoprecipitation that the T allele resulted in increased YY-1 and decreased Nrf-2 binding to the p18 promoter as compared with the C allele in B cells. Thus, YY-1 is a direct regulator of p18 gene expression in an allele-dependent fashion that is consistent with the lupus-associated T allele, inducing a lower p18 transcriptional activity by increasing YY-1 binding. These results establish the p18 -74 C/T mutation as the leading causal variant for the B1a cell expansion that characterizes the NZB and NZM2410 lupus-prone strains.

Antroquinonol differentially modulates T cell activity and reduces interleukin-18 production, but enhances Nrf2 activation, in murine accelerated severe lupus nephritis.

OBJECTIVE: Accelerated severe lupus nephritis (ASLN), with an acute onset of severe clinical manifestations and histopathologic renal lesions, may represent transformation of mild LN to a severe form of glomerulonephritis. Abnormal activation of T and B cells and/or oxidative stress may play a major role in the pathogenesis of ASLN. This study tested the hypothesis that antroquinonol, a purified compound and major effective component of Antrodia camphorata with antiinflammatory and antioxidant activities, might prevent the transformation of mild LN into higher-grade (severe) nephritis in a murine lupus model. METHODS: Experimental ASLN was induced in (NZB×NZW)F1 mice by twice weekly intraperitoneal injections of Salmonella-type lipopolysaccharide (LPS). Starting 2 days after the first dose of LPS, mice were treated daily with antroquinonol, administered by gavage, for different durations up to 5 weeks. RESULTS: Antroquinonol administration significantly ameliorated the proteinuria, hematuria, impairment of renal function, and development of severe renal lesions, especially cellular crescent formation, neutrophil infiltration, fibrinoid necrosis, and T cell proliferation in the glomerulus, as well as periglomerular interstitial inflammation. Mechanistic analyses revealed that antroquinonol 1) inhibited T cell activation/proliferation, but enhanced Treg cell suppression and reduced renal production of interleukin-18 (IL-18); 2) inhibited production of reactive oxygen species and nitric oxide, but increased activation of Nrf2 in the kidney; and 3) suppressed renal inflammation via blocking of NF-κB activation. CONCLUSION: Antroquinonol may have therapeutic potential for the early treatment of ASLN via its differential regulation of T cell function and lowering of IL-18 production, but also via the promotion of Nrf2 activation.

Vaccination of mice for research purpose: alum is as effective as and safer than complete Freund adjuvant.

Systemic lupus erythematosus (SLE) is an autoimmune disease involving many organ systems. Glomerulonephritis (GLN) is one of the major causes of morbidity and mortality in SLE. It has recently been demonstrated that adjuvants of vaccines could cause the so called ASIA syndrome. The study aimed to assess the effects of Complete Freund's Adjuvant (CFA) vs alum injections in NZB/NZWF1 mice. Mice (n=10 each group) were injected with a total volume of 200 µL of: CFA in PBS (group 1), alum in PBS (group 2), PBS (group 3) as controls, PTX3/CFA (group 4), PTX3/alum (group 5), 3 times, 3 weeks apart /given in each injection, three weeks apart from ten weeks of age. Urine samples were collected weekly to evaluate proteinuria. Blood samples were collected before every injection, at 21 weeks of age, and at death to evaluate levels of anti-PTX3 and anti-dsDNA. Proteinuria free survival and survival rates were analyzed by the Kaplan-Meier method using Mantel-Cox's test for comparisons. CFA-treated mice developed both anti-dsDNA antibodies and proteinuria earlier and at higher levels than alumtreated and PBS-injected mice, starting from 13 weeks of age. Proteinuria free survival rates (proteinuria ≥ 300 mg/dL) and survival rates were lower in CFA-treated mice than those treated with alum or injected with PBS (P<0.001 for all). No difference was observed between the alum-treated group and PBS-injected mice. Notably, groups 4 and 5, immunized with PTX3, developed anti-PTX3 antibodies and no significant difference was observed. Alum seems to be as effective as and safer than CFA as adjuvant, since it did not affect disease progression in immunized NZB/NZWF1 mice.

[Experimental study on constitutions of NZB/W F1 lupus mice].

OBJECTIVE: To classify NZB/W F1 lupus mice into three different constitutions, i. e. the cold, normal, and hot constitutions, and to prove the objective existence of the difference among them. METHODS: Using the Four Diagnosis Work Station for Mice (founded by Prof. FANG Zhao-qin, the Research Faculty of Experimental Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine), the body weight, the armpit temperature, the heart rate, the 35-s activities, and the color of their tails and claws (r value) were detected. The total weight was calculated according to the formula: The total weight sum = the correction value of claws x 1 + the correction value of tail x 0.2 + the correction value of the armpit temperature x 0.7 + the correction value of heart rate x0. 05 + the number of the quadrant crossing x 0.025. The NZB/W F1 lupus mice were classified into the three different constitutions according to the higher total weight sum, the hotter the constitution, the lower total weight sum, the colder the constitution. The hydroxyproline content, connective tissue growth factor (CTGF), and transforming growth factor-beta1, (TGF-beta1) gene expression difference in the renal tissue were detected and the immunofluorescence staining observed. RESULTS: Among the 158 NZB/W F1 lupus mice, 33 mice were classified into the hot constitution, 34 into the cold constitution, and 91 into the normal constitution. The renal hydroxyproline content in mice of normal and cold constitutions were higher than that of mice of the hot constitution (P<0.01). No statistical difference was shown between mice of the normal constitution and mice of the cold constitution. The CTGF gene expression level was significantly higher in mice of the cold constitution and mice of the hot constitution than in mice of the normal constitution (P<0.05). No significant difference was found between mice of the cold constitution and mice of the hot constitution. Lower level of TGF-beta1, expression existed in mice of the cold constitution than in mice of the normal constitution or mice of the hot constitution, showing insignificant difference. The immunofluorescence stain of the renal tissue among the three constitutions also showed some difference. CONCLUSION: Constitution difference did exist among NZB/W F1 lupus mice.

Galectin-1-induced down-regulation of T lymphocyte activation protects (NZB x NZW) F1 mice from lupus-like disease.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by a hyperactive immune system, including activation of autoreactive T and B cells. These studies demonstrate that administration of recombinant galectin-1, a ß-galactose binding protein, to SLE-prone (NZB × NZW) F1 mice reduced lymphocyte activation, inhibited serum anti-double-stranded DNA(dsDNA) IgG antibody production, decreased the incidence of proteinuria, and increased survival rate. In addition, recombinant galectin-1'-treated mice had a higher frequency of Foxp3 expression, which suggested an increase in the percentage of peripheral regulatory T cells. Consistent with the finding that there were fewer activated T lymphocytes, ex vivo T cells from mice treated with recombinant galectin-1 exhibited less proliferation in response to TCR stimulation. Furthermore, these cells were less efficient at lipid raft clustering in response to TCR/CD28 engagement, consistent with published reports that galectin-1 can reorganize the synaptic contact to interfere with TCR signaling and activation to prevent T cell activation. Aged galectin-1-deficient mice had higher serum levels of antibodies against dsDNA, elucidating a role for endogenous galectin-1 in decreasing susceptibility to autoimmunity. Together, the findings highlight galectin-1 as a novel potential therapeutic immune modulator for treatment of lupus-like disease.

Experimental study on constitutions of NZB/W F1 lupus mice / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To classify NZB/W F1 lupus mice into three different constitutions, i. e. the cold, normal, and hot constitutions, and to prove the objective existence of the difference among them.</p><p><b>METHODS</b>Using the Four Diagnosis Work Station for Mice (founded by Prof. FANG Zhao-qin, the Research Faculty of Experimental Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine), the body weight, the armpit temperature, the heart rate, the 35-s activities, and the color of their tails and claws (r value) were detected. The total weight was calculated according to the formula: The total weight sum = the correction value of claws x 1 + the correction value of tail x 0.2 + the correction value of the armpit temperature x 0.7 + the correction value of heart rate x0. 05 + the number of the quadrant crossing x 0.025. The NZB/W F1 lupus mice were classified into the three different constitutions according to the higher total weight sum, the hotter the constitution, the lower total weight sum, the colder the constitution. The hydroxyproline content, connective tissue growth factor (CTGF), and transforming growth factor-beta1, (TGF-beta1) gene expression difference in the renal tissue were detected and the immunofluorescence staining observed.</p><p><b>RESULTS</b>Among the 158 NZB/W F1 lupus mice, 33 mice were classified into the hot constitution, 34 into the cold constitution, and 91 into the normal constitution. The renal hydroxyproline content in mice of normal and cold constitutions were higher than that of mice of the hot constitution (P<0.01). No statistical difference was shown between mice of the normal constitution and mice of the cold constitution. The CTGF gene expression level was significantly higher in mice of the cold constitution and mice of the hot constitution than in mice of the normal constitution (P<0.05). No significant difference was found between mice of the cold constitution and mice of the hot constitution. Lower level of TGF-beta1, expression existed in mice of the cold constitution than in mice of the normal constitution or mice of the hot constitution, showing insignificant difference. The immunofluorescence stain of the renal tissue among the three constitutions also showed some difference.</p><p><b>CONCLUSION</b>Constitution difference did exist among NZB/W F1 lupus mice.</p>

Docosahexaenoic acid-enriched fish oil attenuates kidney disease and prolongs median and maximal life span of autoimmune lupus-prone mice.

The therapeutic efficacy of individual components of fish oils (FOs) in various human inflammatory diseases still remains unresolved, possibly due to low levels of n-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) or lower ratio of DHA to EPA. Because FO enriched with DHA (FO-DHA) or EPA (FO-EPA) has become available recently, we investigated their efficacy on survival and inflammatory kidney disease in a well-established animal model of human systemic lupus erythematosus. Results show for the first time that FO-DHA dramatically extends both the median (658 d) and maximal (848 d) life span of (NZB x NZW)F1 (B x W) mice. In contrast, FO-EPA fed mice had a median and maximal life span of approximately 384 and 500 d, respectively. Investigations into possible survival mechanisms revealed that FO-DHA (versus FO-EPA) lowers serum anti-dsDNA Abs, IgG deposition in kidneys, and proteinuria. Further, FO-DHA lowered LPS-mediated increases in serum IL-18 levels and caspase-1-dependent cleavage of pro-IL-18 to mature IL-18 in kidneys. Moreover, FO-DHA suppressed LPS-mediated PI3K, Akt, and NF-kappaB activations in kidney. These data indicate that DHA, but not EPA, is the most potent n-3 fatty acid that suppresses glomerulonephritis and extends life span of systemic lupus erythematosus-prone short-lived B x W mice, possibly via inhibition of IL-18 induction and IL-18-dependent signaling.

RANKL-targeted therapy inhibits bone resorption in experimental Staphylococcus aureus-induced arthritis.

INTRODUCTION: Bacterial arthritis causes rapidly progressing joint destruction in humans. We have shown that addition of bisphosphonates or corticosteroids to conventional antimicrobial agents decreases the activity of osteoclasts, thereby reducing bone destruction. Here we assess the effect of RANKL-targeted treatments using soluble receptor decoy and osteprotegerin (OPG) on the course and outcome of staphylococcal arthritis. METHODS: Treatment was initiated 3 days after Staphylococcus aureus inoculation and included RANK-Fc, OPG-Fc, and OPG-Fc in combination with antibiotics. Control groups were treated with antibiotics, huFc, and PBS. Joints were evaluated for clinical signs of arthritis and histologically for bone and cartilage destruction. Bone mineral density (BMD) was evaluated using a peripheral quantitative computed tomography. Circulating markers of bone metabolism, inflammatory cytokines, and chemokines were analyzed in each group. RESULTS: Mice treated with RANK-Fc or OPG-Fc in combination with antibiotics preserved total BMD and trabecular bone as compared to huFc or antibiotics. Treatment with RANK-Fc or OPG-Fc diminished the levels of bone resorption markers (osteocalcin, CTX-I, and TRACP5b). Neither RANK-Fc nor OPG-Fc influenced significantly the frequency and severity of arthritis. CONCLUSIONS: Inhibition of RANKL signalling efficiently prevents bone loss in the mouse model of bacterial arthritis even when started in the overt phase of infection.

Effects of shikonin isolated from zicao on lupus nephritis in NZB/W F1 mice.

The present study was performed to evaluate the potential protective effects of Shikonin extracted from Zicao on lupus nephritis (LN) using NZB/W F1 mice. Oral administration of Shikonin (24, 40 mg/kg body weight/d) or vehicle was applied to sixty female NZB/W F1 mice of 28-week-old with LN. Treatment with Shikonin for 14 weeks suppressed proteinuria dose-dependently with the mean proteinuria of 274.0 mg/dl and 160.3 mg/dl for low-dose and high-dose Shikonin groups, respectively, compared to 499.2 mg/dl for the vehicle. Also, Shikonin was observed to reduce circulating adhesion molecules significantly and down-regulate intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) mRNA expression in kidney. However, anti-double stranded (ds)DNA antibody in mice with low or high Shikonin dose administration both exhibited no significant elevation, differing from vehicle group. Kidney histological examination showed that renal glomerular lesions were alleviated after Shikonin application. These results suggest that Shikonin has therapeutic effects on LN in NZB/W F1 mice, to which inhibition of anti-dsDNA may be potential contribution, and its part mechanism is related to suppression of mRNA expression of cell adhesion molecules (CAMs) in the kidney.