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1.  Identification of a Common Lupus Disease-Associated microRNA Expression Pattern in Three Different Murine Models of Lupus 
PLoS ONE  2010;5(12):e14302.
Recent reports have shown that microRNAs (miRNAs) regulate vital immunological processes and have emerged as key regulators of immune system development and function. Therefore, it is important to determine miRNA dysregulation and its pathogenic contribution in autoimmune diseases, an aspect not adequately addressed thus far.
Methodology/Principal Findings
In this study, we profiled miRNA expressions in splenic lymphocytes from three murine lupus models (MRL-lpr, B6-lpr and NZB/WF1) with different genetic background by miRNA microarray assays and Real-time RT-PCR. Despite the genetic differences among these three lupus stains, a common set of dysregulated miRNAs (miR-182-96-183 cluster, miR-31, and miR-155) was identified in splenocytes when compared with age-matched control mice. The association of these miRNAs with the disease was highlighted by our observation that this miRNA expression pattern was evident in NZB/W mice only at an age when lupus disease is manifested. Further, we have shown that the miRNA dysregulation in MRL-lpr mice was not simply due to the activation of splenocytes. By Real-time RT-PCR, we confirmed that these miRNAs were upregulated in both purified splenic B and T cells from MRL-lpr mice. miR-127 and miR-379, which were greatly upregulated in splenocytes from lpr mice, were moderately increased in diseased NZB/W mice. In addition, Real-time RT-PCR revealed that miR-146a, miR-101a, and miR-17-92 were also markedly upregulated in splenic T, but not B cells from MRL-lpr mice.
The identification of common lupus disease-associated miRNAs now forms the basis for the further investigation of the pathogenic contribution of these miRNAs in autoimmune lupus, which will advance our knowledge of the role of miRNAs in autoimmunity. Given that miRNAs are conserved, with regard to both evolution and function, our observation of a common lupus disease-associated miRNA expression pattern in murine lupus models is likely to have significant pathogenic, diagnostic, and/or therapeutic implications in human lupus.
PMCID: PMC3000827  PMID: 21170274
2.  Regulation of basement membrane-reactive B cells in BXSB, (NZBxNZW)F1, NZB, and MRL/lpr lupus mice 
Autoimmunity  2013;46(3):188-204.
Autoantibodies to diverse antigens escape regulation in systemic lupus erythematosus under the influence of a multitude of predisposing genes. To gain insight into the differential impact of diverse genetic backgrounds on tolerance mechanisms controlling autoantibody production in lupus, we established a single lupus-derived nephritis associated anti-basement membrane Ig transgene on each of four inbred murine lupus strains, including BXSB, (NZBxNZW)F1, NZB, and MRL/lpr, as approved by the Duke University and the Durham Veterans Affairs Medical Centers’ Animal Care and Use Committees. In nonautoimmune C57BL/6 mice, B cells bearing this anti-laminin Ig transgene are stringently regulated by central deletion, editing, and anergy. Here, we show that tolerance is generally intact in unmanipulated Ig transgenic BXSB, (NZBxNZW)F1, and NZB mice, based on absence of serum transgenic anti-laminin autoantibodies and failure to recover spontaneous anti-laminin monoclonal antibodies. Four- to six-fold depletion of splenic B cells in transgenic mice of these strains, as well as in MRL/lpr transgenic mice, and reduced frequency of IgM+ bone marrow B cells suggest that central deletion is grossly intact. Nonetheless the four strains demonstrate distinct transgenic B cell phenotypes, including endotoxin-stimulated production of anti-laminin antibodies by B cells from transgenic NZB mice, and in vitro hyperproliferation of both endotoxin- and BCR-stimulated B cells from transgenic BXSB mice, which are shown to have an enrichment of CD21-high marginal zone cells. Rare anti-laminin transgenic B cells spontaneously escape tolerance in MRL/lpr mice. Further study of the mechanisms underlying these strain-specific B cell fates will provide insight into genetic modification of humoral autoimmunity in lupus.
PMCID: PMC3625511  PMID: 23157336
autoantibodies; tolerance; transgenic mice; deletion; laminin
3.  Sex differences in the expression of lupus-associated miRNAs in splenocytes from lupus-prone NZB/WF1 mice 
A majority of autoimmune diseases, including systemic lupus erythematosus (SLE), occur predominantly in females. Recent studies have identified specific dysregulated microRNAs (miRNAs) in both human and murine lupus, implying an important contribution of these miRNAs to lupus pathogenesis. However, to date, there is no study that examined sex differences in miRNA expression in immune cells as a plausible basis for sex differences in autoimmune disease. This study addresses this aspect in NZB/WF1 mice, a classical murine lupus model with marked female bias, and further investigates estrogen regulation of lupus-associated miRNAs.
The Taqman miRNA assay system was used to quantify the miRNA expression in splenocytes from male and female NZB/WF1 mice at 17–18, 23, and 30 weeks (wks) of age. To evaluate potential estrogen's effect on lupus-associated miRNAs, 6-wk-old NZB/WF1 male mice were orchidectomized and surgically implanted with empty (placebo) or estrogen implants for 4 and 26 wks, respectively. To assess the lupus status in the NZB/WF1 mice, serum anti-dsDNA autoantibody levels, proteinuria, and renal histological changes were determined.
The sex differences in the expression of lupus-associated miRNAs, including the miR-182-96-183 cluster, miR-155, miR-31, miR-148a, miR-127, and miR-379, were markedly evident after the onset of lupus, especially at 30 wks of age when female NZB/WF1 mice manifested moderate to severe lupus when compared to their male counterparts. Our limited data also suggested that estrogen treatment increased the expression of aforementioned lupus-associated miRNAs, with the exception of miR-155, in orchidectomized male NZB/WF1 mice to a similar level in age-matched intact female NZB/WF1 mice. It is noteworthy that orchiectomy, itself, did not affect the expression of lupus-associated miRNAs.
To our knowledge, this is the first study that demonstrated sex differences in the expression of lupus-associated miRNAs in splenocytes, especially in the context of autoimmunity. The increased expression of lupus-associated miRNA in female NZB/WF1 mice and conceivably in estrogen-treated orchidectomized male NZB/WF1 mice was associated with lupus manifestation. The notable increase of lupus-associated miRNAs in diseased, female NZB/WF1 mice may be a result of both lupus manifestation and the female gender.
PMCID: PMC3843556  PMID: 24175965
Sex differences; Lupus; microRNA; Estrogen; Splenocytes; NZB/WF1
4.  The P2X7 receptor is a candidate product of murine and human lupus susceptibility loci: a hypothesis and comparison of murine allelic products 
Arthritis Research & Therapy  2005;7(3):R468-R475.
Systemic lupus erythematosus and its murine equivalent, modelled in the New Zealand Black and New Zealand White (NZB × NZW)F1 hybrid strain, are polygenic inflammatory diseases, probably reflecting an autoimmune response to debris from cells undergoing programmed cell death. Several human and murine loci contributing to disease have been defined. The present study asks whether the proinflammatory purinergic receptor P2X7, an initiator of a form of programmed cell death known as aponecrosis, is a candidate product of murine and human lupus susceptibility loci. One such locus in (NZB × NZW)F1 mice is lbw3, which is situated at the distal end of NZW chromosome 5. We first assess whether NZB mice and NZW mice carry distinct alleles of the P2RX7 gene as expressed by common laboratory strains, which differ in sensitivity to ATP stimulation. We then compare the responses of NZB lymphocytes, NZW lymphocytes and (NZB × NZW)F1 lymphocytes to P2X7 stimulation. NZB and NZW parental strains express the distinct P2X7-L and P2X7-P alleles of P2RX7, respectively, while lymphocytes from these and (NZB × NZW)F1 mice differ markedly in their responses to P2X7 receptor stimulation. NZB mice and NZW mice express functionally distinct alleles of the proinflammatory receptor, P2X7. We show that current mapping suggests that murine and human P2RX7 receptor genes lie within lupus susceptibility loci lbw3 and SLEB4, and we argue that these encode a product with the functional characteristics consistent with a role in lupus. Furthermore, we argue that aponecrosis as induced by P2X7 is a cell death mechanism with characteristics that potentially have particular relevance to disease pathogenesis.
PMCID: PMC1174943  PMID: 15899033
5.  Induction of a cationic shift in IgG anti-DNA autoantibodies. Role of T helper cells with classical and novel phenotypes in three murine models of lupus nephritis 
The Journal of Experimental Medicine  1987;165(5):1252-1268.
We investigated the underlying mechanisms of systemic autoimmune disease in MRL-+/+, (NZB X NZW)F1, and (NZB X SWR)F1 mice, since these strains develop glomerulonephritis without the superimposition of any secondary lupus-accelerating genes. All three strains manifested a common immunoregulatory defect specific for the production of pathogenic anti-DNA autoantibodies that are of IgG class and cationic in charge. At or just before the age they began to develop lupus nephritis, spleen cells of the mice contained a subpopulation of Th cells that selectively induced their B cells in vitro to produce highly cationic IgG autoantibodies to both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). By contrast, T cells from younger preautoimmune mice were incapable of providing this help. Moreover, only B cells of the older lupus mice could be induced to secrete cationic anti-DNA antibodies of IgG class. B cells of young lupus mice could not produce the cationic autoantibodies even with the help of T cells from the older mice, nor upon stimulation with mitogens. In the older lupus mice we found two sets of Th cells that spontaneously induced the cationic shift in autoantibodies; one set belonged to the classical Th category with L3T4+,Lyt-2- phenotype, whereas the other surprisingly belonged to a double-negative (L3T4-,Lyt-2-), Lyt-1+ subpopulation. The latter set of unusual Th cells were unexpected in these lupus mice since they lacked the lpr (lympho-proliferation) gene. Thus three apparently different murine models of systemic lupus erythematosus possess a common underlying mechanism specific for the spontaneous production of pathogenic anti-DNA autoantibodies.
PMCID: PMC2188317  PMID: 2952749
6.  HSC70 blockade by the therapeutic peptide P140 affects autophagic processes and endogenous MHCII presentation in murine lupus 
Annals of the Rheumatic Diseases  2010;70(5):837-843.
The P140 phosphopeptide issued from the spliceosomal U1-70K small nuclear ribonucleoprotein protein displays protective properties in MRL/lpr lupus-prone mice. It binds both major histocompatibility class II (MHCII) and HSC70/Hsp73 molecules. P140 peptide increases MRL/lpr peripheral blood lymphocyte apoptosis and decreases autoepitope recognition by T cells.
To explore further the mode of action of P140 peptide on HSC70+ antigen-presenting cells.
P140 biodistribution was monitored in real time using an imaging system and by fluorescence and electron microscopy. Fluorescence activated cell sorting and Western blotting experiments were used to evaluate the P140 effects on autophagic flux markers.
P140 fluorescence accumulated especially in the lungs and spleen. P140 peptide reduced the number of peripheral and splenic T and B cells without affecting these cells in normal mice. Remaining MRL/lpr B cells responded normally to mitogens. P140 peptide decreased the expression levels of HSC70/Hsp73 chaperone and stable MHCII dimers, which are both increased in MRL/lpr splenic B cells. It impaired refolding properties of chaperone HSC70. In MRL/lpr B cells, it increased the accumulation of the autophagy markers p62/SQSTM1 and LC3-II, consistent with a downregulated lysosomal degradation during autophagic flux.
The study results suggest that after P140 peptide binding to HSC70, the endogenous (auto)antigen processing might be greatly affected in MRL/lpr antigen-presenting B cells, leading to the observed decrease of autoreactive T-cell priming and signalling via a mechanism involving a lysosomal degradation pathway. This unexpected mechanism might explain the beneficial effect of P140 peptide in treated MRL/lpr mice.
PMCID: PMC3070272  PMID: 21173017
7.  Interplay of autophagy and apoptosis during murine cytomegalovirus infection of RPE cells 
Molecular Vision  2014;20:1161-1173.
Previous studies have demonstrated that autophagy is involved in the pathogenesis of human cytomegalovirus (HCMV) infection. However, whether autophagy is regulated by murine cytomegalovirus (MCMV) infection has not yet been investigated. The purpose of these studies was to determine how autophagy is affected by MCMV infection of the retinal pigment epithelial (RPE) cells and whether there is a functional relationship between autophagy and apoptosis; and if so, how regulation of autophagy impacts apoptosis.
RPE cells were isolated from C57BL/6 mice and infected with MCMV K181. The cells were cultured in medium containing rapamycin, chloroquine, or ammonium chloride. Green fluorescent protein–light chain 3 (GFP-LC3) plasmid was transfected to RPE cells, and the GFP-LC3 positive puncta were counted. Electron microscopic (EM) images were taken to visualize the structure of the autophagic vacuoles. Western blot was performed to detect the expression of related proteins. Trypan blue exclusion assay was used to measure the percentage of viable cells.
Although the LC3B-II levels consistently increased during MCMV infection of RPE cells, administration of chloroquine or ammonium chloride increased LC3B-II expression only at the early stage of infection (6 h post-inoculation [p.i.] and 12 h p.i.), not at or after 24 h p.i. The punctate autophagic vacuoles in the GFP-LC3 transfected RPE cells were counted using light microscopy or by EM examination, the number of autophagic vacuoles was significantly increased in the MCMV-infected RPE cells compared to the uninfected controls. Compared to untreated MCMV-infected control cells, rapamycin treatment resulted in a significant decrease in the cleaved caspase 3 levels as well as a significant decrease in the ratio of phosphorylated mammalian target of rapamycin (mTOR) to total mTOR and in the ratio of phosphorylated P70S6K to total P70S6K. In contrast, chloroquine treatment resulted in a significant increase in the cleaved caspase 3 levels in the MCMV-infected RPE cells.
Autophagic vacuole accumulation was detected during MCMV infection of RPE cells. In contrast, autophagic flux was greatly decreased at or after 24 h p.i. The results suggest that MCMV might have a strategy for inhibiting or blocking autophagy activity by targeting a later autophagy process, such as the formation of autolysosomes or degradation of their content. Our data also suggest that there is a functional relationship between autophagy and apoptosis, which plays an important role during MCMV infection of the RPE.
PMCID: PMC4145064  PMID: 25324684
8.  HRES-1/Rab4-mediated depletion of Drp1 impairs mitochondrial homeostasis and represents a target for treatment in SLE 
Annals of the rheumatic diseases  2013;73(10):1888-1897.
Accumulation of mitochondria underlies T-cell dysfunction in systemic lupus erythematosus (SLE). Mitochondrial turnover involves endosomal traffic regulated by HRES-1/Rab4, a small GTPase that is overexpressed in lupus T cells. Therefore, we investigated whether (1) HRES-1/Rab4 impacts mitochondrial homeostasis and (2) Rab geranylgeranyl transferase inhibitor 3-PEHPC blocks mitochondrial accumulation in T cells, autoimmunity and disease development in lupus-prone mice.
Mitochondria were evaluated in peripheral blood lymphocytes (PBL) of 38 SLE patients and 21 healthy controls and mouse models by flow cytometry, microscopy and western blot. MRL/lpr mice were treated with 125 μg/kg 3-PEHPC or 1 mg/kg rapamycin for 10 weeks, from 4 weeks of age. Disease was monitored by antinuclear antibody (ANA) production, proteinuria, and renal histology.
Overexpression of HRES-1/Rab4 increased the mitochondrial mass of PBL (1.4-fold; p=0.019) and Jurkat cells (2-fold; p=0.000016) and depleted the mitophagy initiator protein Drp1 both in human (−49%; p=0.01) and mouse lymphocytes (−41%; p=0.03). Drp1 protein levels were profoundly diminished in PBL of SLE patients (−86±3%; p=0.012). T cells of 4-week-old MRL/lpr mice exhibited 4.7-fold over-expression of Rab4A (p=0.0002), the murine homologue of HRES-1/ Rab4, and depletion of Drp1 that preceded the accumulation of mitochondria, ANA production and nephritis. 3-PEHPC increased Drp1 (p=0.03) and reduced mitochondrial mass in T cells (p=0.02) and diminished ANA production (p=0.021), proteinuria (p=0.00004), and nephritis scores of lupus-prone mice (p<0.001).
These data reveal a pathogenic role for HRES-1/Rab4-mediated Drp1 depletion and identify endocytic control of mitophagy as a treatment target in SLE.
PMCID: PMC4047212  PMID: 23897774
9.  HRES-1/Rab4-mediated depletion of Drp1 impairs mitochondrial homeostasis and represents a target for treatment in SLE 
Annals of the Rheumatic Diseases  2013;73(10):1888-1897.
Accumulation of mitochondria underlies T-cell dysfunction in systemic lupus erythematosus (SLE). Mitochondrial turnover involves endosomal traffic regulated by HRES-1/Rab4, a small GTPase that is overexpressed in lupus T cells. Therefore, we investigated whether (1) HRES-1/Rab4 impacts mitochondrial homeostasis and (2) Rab geranylgeranyl transferase inhibitor 3-PEHPC blocks mitochondrial accumulation in T cells, autoimmunity and disease development in lupus-prone mice.
Mitochondria were evaluated in peripheral blood lymphocytes (PBL) of 38 SLE patients and 21 healthy controls and mouse models by flow cytometry, microscopy and western blot. MRL/lpr mice were treated with 125 μg/kg 3-PEHPC or 1 mg/kg rapamycin for 10 weeks, from 4 weeks of age. Disease was monitored by antinuclear antibody (ANA) production, proteinuria, and renal histology.
Overexpression of HRES-1/Rab4 increased the mitochondrial mass of PBL (1.4-fold; p=0.019) and Jurkat cells (2-fold; p=0.000016) and depleted the mitophagy initiator protein Drp1 both in human (−49%; p=0.01) and mouse lymphocytes (−41%; p=0.03). Drp1 protein levels were profoundly diminished in PBL of SLE patients (−86±3%; p=0.012). T cells of 4-week-old MRL/lpr mice exhibited 4.7-fold over-expression of Rab4A (p=0.0002), the murine homologue of HRES-1/Rab4, and depletion of Drp1 that preceded the accumulation of mitochondria, ANA production and nephritis. 3-PEHPC increased Drp1 (p=0.03) and reduced mitochondrial mass in T cells (p=0.02) and diminished ANA production (p=0.021), proteinuria (p=0.00004), and nephritis scores of lupus-prone mice (p<0.001).
These data reveal a pathogenic role for HRES-1/Rab4-mediated Drp1 depletion and identify endocytic control of mitophagy as a treatment target in SLE.
PMCID: PMC4047212  PMID: 23897774
Autoimmune Diseases; Autoimmunity; Systemic Lupus Erythematosus
10.  Autophagic flux determines cell death and survival in response to Apo2L/TRAIL (dulanermin) 
Molecular Cancer  2014;13:70.
Macroautophagy is a catabolic process that can mediate cell death or survival. Apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment (TR) is known to induce autophagy. Here we investigated whether SQSTM1/p62 (p62) overexpression, as a marker of autophagic flux, was related to aggressiveness of human prostate cancer (PCa) and whether autophagy regulated the treatment response in sensitive but not resistant PCa cell lines.
Immunostaining and immunoblotting analyses of the autophagic markers p62 [in PCa tissue microarrays (TMAs) and PCa cell lines] and LC3 (in PCa cell lines), transmission electron microscopy, and GFP-mCherry-LC3 were used to study autophagy induction and flux. The effect of autophagy inhibition using pharmacologic (3-methyladenine and chloroquine) and genetic [(short hairpin (sh)-mediated knock-down of ATG7 and LAMP2) and small interfering (si)RNA-mediated BECN1 knock-down] approaches on TR-induced cell death was assessed by clonogenic survival, sub-G1 DNA content, and annexinV/PI staining by flow cytometry. Caspase-8 activation was determined by immunoblotting.
We found that increased cytoplasmic expression of p62 was associated with high-grade PCa, indicating that autophagy signaling might be important for survival in high-grade tumors. TR-resistant cells exhibited high autophagic flux, with more efficient clearance of p62-aggregates in four TR-resistant PCa cell lines: C4-2, LNCaP, DU145, and CWRv22.1. In contrast, autophagic flux was low in TR-sensitive PC3 cells, leading to accumulation of p62-aggregates. Pharmacologic (chloroquine or 3-methyladenine) and genetic (shATG7 or shLAMP2) inhibition of autophagy led to cell death in TR-resistant C4-2 cells. shATG7-expressing PC3 cells, were less sensitive to TR-induced cell death whereas those shLAMP2-expressing were as sensitive as shControl-expressing PC3 cells. Inhibition of autophagic flux using chloroquine prevented clearance of p62 aggregates, leading to caspase-8 activation and cell death in C4-2 cells. In PC3 cells, inhibition of autophagy induction prevented p62 accumulation and hence caspase-8 activation.
We show that p62 overexpression correlates with advanced stage human PCa. Pharmacologic and genetic inhibition of autophagy in PCa cell lines indicate that autophagic flux can determine the cellular response to TR by regulating caspase-8 activation. Thus, combining various autophagic inhibitors may have a differential impact on TR-induced cell death.
PMCID: PMC3998041  PMID: 24655592
Autophagy; p62/SQSTM1; Caspase-8; Prostate cancer; Apo2L/TRAIL; Dulanermin
11.  B cell dependence on and response to accessory signals in murine lupus strains 
The Journal of Experimental Medicine  1983;157(6):1815-1827.
B cell hyperactivity, a feature common to all lupus-prone murine strains, may be caused by hyperresponsiveness to, overproduction of, or bypassing of certain signals required for B cell activation, proliferation, and differentiation. In this study, we have compared the responses of B cells from three lupus-prone strains of mice (BXSB males, MRL and NZB/W females) and normal strains in a number of assays for which two or more signals are required to obtain a response. In medium to low density cultures of B cells from BXSB and NZB/W but not MRL/l lupus mice, the cells' proliferation induced by bacterial lipopolysaccharide (LPS) or anti-mu antibody was much higher than that of B cells from normal controls. At low B cell density, polyclonal activation by these substances and subsequent Ig secretion were dependent on accessory signals present in supernatants of concanavalin A-treated normal lymphocytes (CAS) or on the MRL/l proliferating T cell- derived B cell differentiation factor (L-BCDF) in both lupus-prone and immunologically normal mice. However, the responses of B cells from BXSB and NZB/W, but not MRL/l, mice to these accessory signals were higher than those of normal mice. Ig synthesis by fresh B cells of BXSB and NZB/W mice cultured in the absence of mitogens but in the presence of CAS or L-BCDF was higher than by similar cells from other strains, suggesting an increased frequency of B cells activated in vivo in these two autoimmune strains of mice. The patterns of IgG subclass secretion in response to LPS (without added CAS or L-BCDF) were abnormal in all lupus strains, with a predominance of IgG2b and/or IgG2a and low levels of IgG3, contrary to normal B cells for which IgG3 synthesis predominated. However, IgG1 synthesis in vitro by autoimmune and normal B cells alike was highly dependent on T cell-derived soluble mediators. Antigen-specific responses to SRBC in vitro of B cells from all lupus strains, like those of B cells from normal strains, required a minimum of three signals (antigen, LPS, T cell-derived antigen nonspecific helper factors). Yet, once triggered, B cells of BXSB and NZB/W mice gave higher responses than those of the other strains. We conclude that B cells of lupus mice have signal requirements similar to those of normal mice. Nevertheless, B cells of BXSB and NZB/W, but not MRL/l, lupus mice hyperrespond or process some accessory signals abnormally.
PMCID: PMC2187057  PMID: 6406639
12.  Effects of altered prenatal hormonal environment on expression of autoimmune disease in NZB/NZW mice. 
Environmental Health Perspectives  1996;104(Suppl 4):815-821.
F1 hybrid New Zealand Black (NZB) x New Zealand White (NZM) (NZB/NZW) mice spontaneously develop an autoimmune disease analogous to systemic lupus erythematosus (SLE). Testosterone experts a powerful suppressive effect on this disorder in adult NZB/NZW mice. A series of experiments was designed to determine if disease would also be suppressed by exposing fetal NZB/NZW mice to increased testosterone. A model was developed in which NZB dams carrying NZB/NZW fetuses were treated with testosterone in a dose adequate to masculinize the external genitalia in female fetuses. NZB/NZW mice that were derived from testosterone-treated dams and control NZB/NZW offspring were followed in a longevity study and had serial assays to assess development of SLE. Additional experiments were carried out to measure lymphocyte subsets and responses to mitogens. Results were compared with F1 hybrid offspring of C57BL/6 dams crossed with DBA/2 males, which are not autoimmune and do not develop SLE. Spleen cells from these groups were tested for Thy 1.2, CD4, CD8, and IgM receptors, and for responses to the mitogens Concanavalin A (ConA) and lipopolysaccharide. Control male NZB/NZW fetuses had unexpectedly high serum estradiol, which decreased significantly with maternal testosterone treatment. The testosterone-exposed male NZB/NZW fetuses developed into adults that lived longer than male NZB/NZW controls. Testosterone treatment of the dam was associated with elevated terminal anti-DNA levels but did not alter markers of renal diseases in adult NZB/NZW mice of either sex. Testosterone-exposed NZB/NZW females had altered T-lymphocyte subsets and testosterone-exposed males had increased response to ConA compared to controls. In male NZB/NZW fetuses whose mothers were administered testosterone, the naturally high level of circulating estradiol observed in untreated male fetuses was decreased significantly. This decrease was associated with an increase in longevity. This unique observation has important implications for fetal exposure to endocrine disruptors in the environment.
PMCID: PMC1469666  PMID: 8880004
13.  Protective and Pathogenic Roles for B Cells During Systemic Autoimmunity in NZB/W F1 Mice1 
Delineating the relative contributions of B lymphocytes during the course of autoimmune disease has been difficult. Therefore, the effects of depleting all mature B cells using a potent CD20 mAb, or depleting circulating and marginal zone B cells using a ligand-blocking CD22 mAb were compared in NZB/W F1 mice, a model for human systemic lupus erythematosus. Single low-dose mAb treatments depleted B cells efficiently in both NZB/W F1 and C57BL/6 mice. Prophylactic B cell depletion by repeated CD20 mAb treatments prolonged survival during pristane-accelerated lupus in NZB/W F1 mice, while CD22 mAb had little effect. Despite effective B cell depletion, neither mAb treatment prevented autoantibody generation. In addition, CD20, CD22, and control mAb-treated NZB/W F1 mice developed anti-mouse IgG autoantibodies in contrast to parental NZB and NZW strains, which may have reduced the effectiveness of B cell depletion. Despite this, low dose CD20 mAb treatment initiated at 12–28 weeks of age and then every 4 weeks significantly delayed spontaneous disease in NZB/W F1 mice. By contrast, B cell depletion starting at 4 wks of age hastened disease onset, which paralleled depletion of the IL-10-producing regulatory B cell subset called B10 cells. B10 cells were phenotypically similar in NZB/W F1 and C57BL/6 mice, but were expanded significantly in young NZB/W F1 mice. Thus, B cell depletion had significant effects on NZB/W mouse survival that were dependent on the timing of treatment initiation. Thereby, distinct B cell populations may have opposing protective and pathogenic roles during lupus progression.
PMCID: PMC3734557  PMID: 20368280
B cells; CD20; CD22; immunotherapy; lupus; regulatory B cells
14.  Sepsis Induces Extensive Autophagic Vacuolization in Hepatocytes –a clinical and laboratory based study 
Autophagy is the regulated process cells use to recycle non-essential, redundant, or inefficient components and is an adaptive response during times of stress. In addition to its role in enabling the cell to gain vital nutrients in times of stress, autophagy can also be involved in elimination of intracellular microorganisms, tumor suppression, and antigen presentation. Because of difficulty in diagnosing autophagy, few clinical studies have been performed. This study examined whether autophagy occurs in hepatocytes during sepsis. Electron microscopy (EM) was performed on liver samples obtained from both an observational clinical cohort of 6 septic patients and 4 control patients as well as liver specimens from mice with surgical sepsis (via cecal ligation and puncture (CLP)) or sham operation. EM demonstrated increased autophagic vacuoles in septic versus non-septic patients. Randomly selected fields (3,000 square microns) from control and septic patients contained 1.2 ± 1.5 versus 5.3 ± 3.3 (mean ± SD) complex lysosomal/autophagolysosomal structures per image respectively (P<0.001). In rare instances, hepatocytes with autophagic vacuoles appeared to be unequivocally committed to death. Membrane alterations (membrane vacuoles, invagination into adjacent organelles and myelin figure-like changes) occur in a subpopulation of mitochondria in sepsis, but other hepatocyte organelles showed no consistent ultrastructural injury. Findings in murine sepsis paralleled those of patients, with 7.2 ± 1.9 versus 38.7 ± 3.9 lysosomal/autophagolysosomal structures in sham and septic mice, respectively (P =0.002). Quantitative RT-PCR demonstrated that sepsis-induced the upregulation of select apoptosis and cytokine gene expression with minimal changes in the core autophagy genes in liver. In conclusion, hepatocyte autophagic vacuolization increases during sepsis and is associated with mitochondrial injury. However, it is not possible to determine whether the increase in autophagic vacuolization is an adaptive response or a harbinger of cell death.
PMCID: PMC3822608  PMID: 19188912
cell death; inflammation; cytokines; electron microscopy; gene expression
15.  Dual B Cell Immunotherapy Is Superior to Individual Anti-CD20 Depletion or BAFF Blockade in Murine Models of Spontaneous or Accelerated Lupus 
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.
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.
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.
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.
PMCID: PMC4312898  PMID: 25303150
16.  Interferon Regulatory Factor-5 Deficiency Ameliorates Disease Severity in the MRL/lpr Mouse Model of Lupus in the Absence of a Mutation in DOCK2 
PLoS ONE  2014;9(7):e103478.
Interferon regulatory factor 5 (IRF5) polymorphisms are strongly associated with an increased risk of developing the autoimmune disease systemic lupus erythematosus. In mouse lupus models, IRF5-deficiency was shown to reduce disease severity consistent with an important role for IRF5 in disease pathogenesis. However these mouse studies were confounded by the recent demonstration that the IRF5 knockout mouse line contained a loss-of-function mutation in the dedicator of cytokinesis 2 (DOCK2) gene. As DOCK2 regulates lymphocyte trafficking and Toll-like receptor signaling, this raised the possibility that some of the protective effects attributed to IRF5 deficiency in the mouse lupus models may instead have been due to DOCK2 deficiency. We have therefore here evaluated the effect of IRF5-deficiency in the MRL/lpr mouse lupus model in the absence of the DOCK2 mutation. We find that IRF5-deficient (IRF5−/−) MRL/lpr mice develop much less severe disease than their IRF5-sufficient (IRF5+/+) littermates. Despite markedly lower serum levels of anti-nuclear autoantibodies and reduced total splenocyte and CD4+ T cell numbers, IRF5−/− MRL/lpr mice have similar numbers of all splenic B cell subsets compared to IRF5+/+ MRL/lpr mice, suggesting that IRF5 is not involved in B cell development up to the mature B cell stage. However, IRF5−/− MRL/lpr mice have greatly reduced numbers of spleen plasmablasts and bone marrow plasma cells. Serum levels of B lymphocyte stimulator (BLyS) were markedly elevated in the MRL/lpr mice but no effect of IRF5 on serum BLyS levels was seen. Overall our data demonstrate that IRF5 contributes to disease pathogenesis in the MRL/lpr lupus model and that this is due, at least in part, to the role of IRF5 in plasma cell formation. Our data also suggest that combined therapy targeting both IRF5 and BLyS might be a particularly effective therapeutic approach in lupus.
PMCID: PMC4116215  PMID: 25076492
17.  IL-21 Receptor Is Required for the Systemic Accumulation of Activated B and T Lymphocytes in MRL/MpJ-Faslpr/lpr/J Mice 
MRL/MpJ-Faslpr/lpr/J (MRLlpr) mice develop lupus-like disease manifestations in an IL-21–dependent manner. IL-21 is a pleio-tropic cytokine that can influence the activation, differentiation, and expansion of B and T cell effector subsets. Notably, auto-reactive CD4+ T and B cells spontaneously accumulate in MRLlpr mice and mediate disease pathogenesis. We sought to identify the particular lymphocyte effector subsets regulated by IL-21 in the context of systemic autoimmunity and, thus, generated MRLlpr mice deficient in IL-21R (MRLlpr.IL-21R−/−). Lymphadenopathy and splenomegaly, which are characteristic traits of the MRLlpr model were significantly reduced in the absence of IL-21R, suggesting that immune activation was likewise decreased. Indeed, spontaneous germinal center formation and plasma cell accumulation were absent in IL-21R–deficient MRLlpr mice. Correspondingly, we observed a significant reduction in autoantibody titers. Activated CD4+ CD44+ CD62Llo T cells also failed to accumulate, and CD4+ Th cell differentiation was impaired, as evidenced by a significant reduction in CD4+ T cells that produced the pronephritogenic cytokine IFN-γ. T extrafollicular helper cells are a recently described subset of activated CD4+ T cells that function as the primary inducers of autoantibody production in MRLlpr mice. Importantly, we demonstrated that T extrafollicular helper cells are dependent on IL-21R for their generation. Together, our data highlighted the novel observation that IL-21 is a critical regulator of multiple pathogenic B and T cell effector subsets in MRLlpr mice.
PMCID: PMC3618484  PMID: 22231702
18.  Suppression of Skin and Kidney Disease by Inhibition of Spleen Tyrosine Kinase in Lupus-Prone Mice 
Arthritis and rheumatism  2010;62(7):2086-2092.
Spleen tyrosine kinase (Syk) is involved in membrane-mediated signaling in various cells, including immune cells. It is overexpressed in T cells from patients with systemic lupus erythematosus (SLE), and its inhibition has been shown to improve T cell function as well as to improve disease manifestations in (NZB × NZW)F1 lupus-prone mice and in patients with rheumatoid arthritis. While clinical trials examining Syk inhibition in patients with SLE are being considered, the aim of our experiments was to determine whether the therapeutic effects of Syk inhibition extend to other strains of lupus-prone mice and whether they result in improvement in skin disease and modification of established disease.
Female MRL/lpr or BAK/BAX mice were studied. Starting either at age 4 weeks (before disease) or at age 16 weeks (after established disease) and continuing for up to 16 weeks, mice were fed chow containing the Syk inhibitor R788 or control chow.
We found that inhibition of Syk in MRL/lpr and BAK/BAX mice prevented the development of skin disease and significantly reduced established skin disease. Similarly, Syk inhibition reduced the size of the spleen and lymph nodes, suppressed the development of renal disease, and suppressed established renal disease. Discontinuation of treatment resulted in extended suppression of skin disease for at least 8 weeks and suppression of renal disease for 4 weeks.
Syk inhibition suppresses the development of lupus skin and kidney disease in lupus-prone mice, suppresses established disease in lupus-prone mice, and may represent a valuable treatment for patients with SLE.
PMCID: PMC2902591  PMID: 20222110
19.  The SLAM family member CD48 (Slamf2) protects lupus-prone mice from autoimmune nephritis 
Journal of autoimmunity  2011;37(1):48-57.
Polymorphisms in the SLAM family of leukocyte cell surface regulatory molecules have been associated with lupus-like phenotypes in both humans and mice. The murine Slamf gene cluster lies within the lupus-associated Sle1b region of mouse chromosome 1. Non-autoreactive C57BL/6 (B6) mice that have had this region replaced by syntenic segments from other mouse strains (i.e. 129, NZB and NZW) are B6 congenic strains that spontaneously produce non-nephritogenic lupus-like autoantibodies. We have recently reported that genetic ablation of the SLAM family member CD48 (Slamf2) drives full-blown autoimmune disease with severe proliferative glomerulonephritis (CD48GN) in B6 mice carrying 129 sequences of the Sle1b region (B6.129CD48-/-). We also discovered that BALB/c mice with the same 129-derived CD48-null allele (BALB.129CD48-/-) have neither nephritis nor anti-DNA autoantibodies, indicating that strain specific background genes modulate the effects of CD48 deficiency. Here we further examine this novel model of lupus nephritis in which CD48 deficiency transforms benign autoreactivity into fatal nephritis. CD48GN is characterized by glomerular hypertrophy with mesangial expansion, proliferation and leukocytic infiltration. Immune complexes deposit in mesangium and in sub-endothelial, sub-epithelial and intramembranous sites along the glomerular basement membrane. Afflicted mice have low grade proteinuria, intermittent hematuria and their progressive renal injury manifests with elevated urine NGAL levels and with uremia. In contrast to the lupus-like B6.129CD48-/- animals, neither BALB.129CD48-/- mice nor B6 × BALB/c F1.129CD48-/- progeny have autoimmune traits, indicating that B6-specific background genes modulate the effect of CD48 on lupus nephritis in a recessive manner.
PMCID: PMC3110600  PMID: 21561736
CD48(Slamf2); lupus nephritis; anti-DNA autoantibodies; systemic lupus erythematosus; murine lupus; Sle1b
20.  Suppression of Glomerulonephritis in NZB/NZW Lupus Prone Mice by Adoptive Transfer of Ex Vivo Expanded Regulatory T Cells 
PLoS ONE  2009;4(6):e6031.
Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown cause characterized by expansion of autoreactive lymphocytes. Regulatory T cells (Tregs) are a component of the normal immune system and contribute to the maintenance of peripheral tolerance. Treg abnormalities have been associated with several autoimmune diseases and there is interest in the role of Tregs in SLE. We previously demonstrated that transfer of expanded CD4+CD25+CD62LHI Tregs slows the development of lupus in (NZBxNZW)F1 (B/W) mice. However in the absence of Treg specific surface antigens, cell purification remains a compromise between the breadth and purity of the population isolated. Importantly, purified populations always contain Foxp3− effector T cells (Teffs) that theoretically could exacerbate autoimmunity in the recipient. Here we explore the impact of transferring the more comprehensive, but less pure Treg subset defined by CD4+CD25+ expression on development of murine lupus. All cells were FACS sorted and expanded prior to adoptive transfer. Development of proteinuria and survival were measured. We found that exogenous expansion of CD4+CD25+ cells produced a population containing 70–85% CD4+Foxp3+Tregs. Expanded Tregs had higher CTLA-4 and Foxp3 expression, increased in vitro suppression capacity, and prolonged in vivo survival as compared to freshly isolated cells. Adoptive transfer of expanded CD4+CD25+ Tregs inhibited the onset of glomerulonephritis and prolonged survival in mice. Importantly the population of Teff contained within the adoptively transferred cells had reduced survival and proliferation capacity as compared to either co-transferred Tregs or transferred Teffs expanded in the absence of Tregs. These studies demonstrate that adoptive transfer of expanded CD4+CD25+Foxp3+Tregs has the capacity to inhibit the onset of murine lupus and that this capacity is significant despite transfer of co-cultured Teff cells. These data indicate that when co-expanded with regulatory T cells, exogenously activated Teffs from autoimmune patients may not pose a significant risk of promoting disease.
PMCID: PMC2696596  PMID: 19551149
21.  Disease Progression in MRL/lpr Lupus-Prone Mice Is Reduced by NCS 613, a Specific Cyclic Nucleotide Phosphodiesterase Type 4 (PDE4) Inhibitor 
PLoS ONE  2012;7(1):e28899.
Systemic lupus erythematosus is a polymorphic and multigenic inflammatory autoimmune disease. Cyclic AMP (cAMP) modulates inflammation and the inhibition of cyclic nucleotide phosphodiesterase type 4 (PDE4), which specifically hydrolyzes cAMP, inhibits TNFα secretion. This study was aimed at investigating the evolution of PDE activity and expression levels during the course of the disease in MRL/lpr lupus-prone mice, and to evaluate in these mice the biological and clinical effects of treatments with pentoxifylline, denbufylline and NCS 613 PDE inhibitors. This study reveals that compared to CBA/J control mice, kidney PDE4 activity of MRL/lpr mice increases with the disease progression. Furthermore, it showed that the most potent and selective PDE4 inhibitor NCS 613 is also the most effective molecule in decreasing proteinuria and increasing survival rate of MRL/lpr mice. NCS 613 is a potent inhibitor, which is more selective for the PDE4C subtype (IC50 = 1.4 nM) than the other subtypes (PDE4A, IC50 = 44 nM; PDE4B, IC50 = 48 nM; and PDE4D, IC50 = 14 nM). Interestingly, its affinity for the High Affinity Rolipram Binding Site is relatively low (Ki = 148 nM) in comparison to rolipram (Ki = 3 nM). Finally, as also observed using MRL/lpr peripheral blood lymphocytes (PBLs), NCS 613 inhibits basal and LPS-induced TNFα secretion from PBLs of lupus patients, suggesting a therapeutic potential of NCS 613 in systemic lupus. This study reveals that PDE4 represent a potential therapeutic target in lupus disease.
PMCID: PMC3256138  PMID: 22247763
22.  ICOS Controls Effector Function but Not Trafficking Receptor Expression of Kidney-Infiltrating Effector T Cells in Murine Lupus1 
Renal pathology in systemic lupus erythematosus involves both autoantibody deposition and a cellular inflammatory response, both of which are mediated by effector CD4 T cells. MRLlpr mice spontaneously develop massive perivascular infiltrates, but the pathways that regulate the development, trafficking, and effector functions of kidney-infiltrating T cells are poorly defined. To address these questions, we first surveyed inflammatory chemokine protein levels in nephritic kidneys from lupus-prone MRLlpr mice. After identifying highly elevated levels of the CXCR3 ligand CXCL9, we found that kidney-infiltrating effectors are enriched for expression of CXCR3, as well as P-selectin ligand and ICOS. Using genetic ablation, we demonstrate that ICOS plays an essential role in the establishment of renal perivascular infiltrates, although a small number of infiltrating cells remain around the blood vessels. Interestingly, though IgG autoantibody production is substantially reduced in Icos−/− MRLlpr mice, the progression of immune complex glomerulonephritis is only modestly diminished and the production of inflammatory chemokines, such as CXCL9, remains high in the kidney. We find that Icos−/− effector cell numbers are only slightly reduced and these have normal expression of CXCR3 and P-selectin ligand with intact migration to CXCL9. However, they have impaired production of inflammatory cytokines and fail to show evidence of efficient proliferation in the kidney. Thus, while dispensable for acquisition of renal trafficking receptor expression, ICOS is strictly required for local inflammatory functions of autoreactive CD4 T cells in murine lupus.
PMCID: PMC2746004  PMID: 19299705
23.  Association of FKBP51 with Priming of Autophagy Pathways and Mediation of Antidepressant Treatment Response: Evidence in Cells, Mice, and Humans 
PLoS Medicine  2014;11(11):e1001755.
Theo Rein and colleagues examine the role of FKBP51 in the actions of antidepressants, with a particular focus on pathways of autophagy.
Please see later in the article for the Editors' Summary
FK506 binding protein 51 (FKBP51) is an Hsp90 co-chaperone and regulator of the glucocorticoid receptor, and consequently of stress physiology. Clinical studies suggest a genetic link between FKBP51 and antidepressant response in mood disorders; however, the underlying mechanisms remain elusive. The objective of this study was to elucidate the role of FKBP51 in the actions of antidepressants, with a particular focus on pathways of autophagy.
Methods and Findings
Established cell lines, primary neural cells, human blood cells of healthy individuals and patients with depression, and mice were treated with antidepressants. Mice were tested for several neuroendocrine and behavioral parameters. Protein interactions and autophagic pathway activity were mainly evaluated by co-immunoprecipitation and Western blots. We first show that the effects of acute antidepressant treatment on behavior are abolished in FKBP51 knockout (51KO) mice. Autophagic markers, such as the autophagy initiator Beclin1, were increased following acute antidepressant treatment in brains from wild-type, but not 51KO, animals. FKBP51 binds to Beclin1, changes decisive protein interactions and phosphorylation of Beclin1, and triggers autophagic pathways. Antidepressants and FKBP51 exhibited synergistic effects on these pathways. Using chronic social defeat as a depression-relevant stress model in combination with chronic paroxetine (PAR) treatment revealed that the stress response, as well as the effects of antidepressants on behavior and autophagic markers, depends on FKBP51. In human blood cells of healthy individuals, FKBP51 levels correlated with the potential of antidepressants to induce autophagic pathways.
Importantly, the clinical antidepressant response of patients with depression (n = 51) could be predicted by the antidepressant response of autophagic markers in patient-derived peripheral blood lymphocytes cultivated and treated ex vivo (Beclin1/amitriptyline: r = 0.572, p = 0.003; Beclin1/PAR: r = 0.569, p = 0.004; Beclin1/fluoxetine: r = 0.454, p = 0.026; pAkt/amitriptyline: r = −0.416, p = 0.006; pAkt/PAR: r = −0.355, p = 0.021; LC3B-II/PAR: r = 0.453, p = 0.02), as well as by the lymphocytic expression levels of FKBP51 (r = 0.631, p<0.0001), pAkt (r = −0.515, p = 0.003), and Beclin1 (r = 0.521, p = 0.002) at admission. Limitations of the study include the use of male mice only and the relatively low number of patients for protein analyses.
To our knowledge, these findings provide the first evidence for the molecular mechanism of FKBP51 in priming autophagic pathways; this process is linked to the potency of at least some antidepressants. These newly discovered functions of FKBP51 also provide novel predictive markers for treatment outcome, consistent with physiological and potential clinical relevance.
Please see later in the article for the Editors' Summary
Editors' Summary
Everyone feels miserable sometimes, but about one in six people will have an episode of clinical depression during their lifetime. For people who are clinically depressed, overwhelming feelings of sadness, anxiety, and hopelessness can last for months or years. Affected individuals lose interest in activities they used to enjoy, they sometimes have physical symptoms such as disturbed sleep, and they may contemplate suicide. Clinicians diagnose depression and determine its severity using questionnaires (“depression rating scales”) that explore the patient's feelings and symptoms. Mild depression is often treated with talking therapies (psychotherapy) such as cognitive behavioral therapy, which helps people change negative ways of thinking. For more severe depression, patients are also usually prescribed an antidepressant, most commonly a “selective serotonin reuptake inhibitor” such as paroxetine or a tricyclic antidepressant such as amitriptyline.
Why Was This Study Done?
Unfortunately, antidepressants don't work for more than half of patients. Moreover, because it is unclear how antidepressants work, it is not possible to predict which patients will respond to which antidepressants. Thus, matching patient to drug can be a lengthy, sometimes unsuccessful, process. Here, the researchers use several approaches to test the hypothesis that a protein called FK506 binding protein 51 (FKBP51) is involved in the actions of antidepressants and to investigate whether the ability of both FKBP51 and antidepressants to regulate a process called autophagy underlies the impact of FKBP51 on antidepressant responses. FKBP51 is a regulator of stress physiology, which is connected to the development and treatment of depression; genetic studies have suggested a link between FKBP51 expression and the antidepressant response rate. Some antidepressants are known to alter the initial steps in the autophagy pathway, a multistep process that maintains the integrity of cells through regulated degradation and recycling of cellular components; however, the potential synergistic role of FKBP51 and antidepressants in regulating pathways of autophagy are unknown.
What Did the Researchers Do and Find?
The researchers first treated wild-type mice and FKBP51 knockout mice (genetically altered animals that make no FKBP51) with an acute dose of antidepressant and compared their behavior in a forced swim test, an assay that measures the action of antidepressants in mice by determining how long the mice struggle or float inertly when placed in deep water. As expected, acute antidepressant treatment increased the time that wild-type mice spent struggling. However, this effect of antidepressant treatment was greatly attenuated in the FKBP51 knockout mice. Moreover, the levels of several autophagy markers increased in the brains of wild-type mice following antidepressant treatment but not in the brains of FKBP51 knockout mice. Next, using “chronic social defeat stress” to model the “endophenotype” of depression (a combination of physiological, hormonal, and behavioral traits seen in people with depression) in mice, the researchers showed that the stress response and the effect of chronic antidepressants on behavior and on autophagic markers all depend on FKBP51. Using cell-based assays, the researchers showed that antidepressants and FKBP51 had synergistic (interactive) effects on the autophagic pathway and that, in human blood cells, FKBP51 levels correlated with the potential of antidepressants to induce autophagic pathways. Finally, the researchers report that the clinical response to antidepressant treatment in 51 patients with depression was associated with the response of autophagic markers in their peripheral blood lymphocytes to antidepressant treatment in test tubes, and that the expression levels of FKBP51 and autophagy markers in patient lymphocytes at admission were associated with subsequent clinical responses to antidepressants.
What Do These Findings Mean?
These findings suggest that the protein FKBP51 is required for the effects of both acute and chronic treatment with some antidepressants on behavior and on autophagic pathways in mice. These findings also reveal an association between antidepressant treatment responses in patients and both the expression levels of FKBP51 and autophagy markers in lymphocytes at admission and the response of autophagic markers to antidepressant treatment in patient lymphocytes. The accuracy of these findings is limited by the small number of clinical samples available for analysis, by the use of only male mice in the animal experiments, and by the inability of animal models of depression to fully replicate the human condition. Nevertheless, these findings identify the early stages of autophagy as potential targets for the development of new antidepressants and identify several potential biomarkers that might, after further clinical validation, help clinicians predict antidepressant efficacy in patients with depression.
Additional Information
Please access these websites via the online version of this summary at
The US National Institute of Mental Health provides information on all aspects of depression (in English and Spanish), including information on antidepressants
The UK National Health Service Choices website provides detailed information about depression and about antidepressants; it also provides personal stories about depression
The UK charity Mind provides information on depression, including some personal stories about depression
More personal stories about depression are available from
MedlinePlus provides links to other resources about depression
Wikipedia has a page on autophagy (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
The patients included in this study were all enrolled in the Munich Antidepressant Response Signature project, which aims to identify gene variants and biomarkers that predict treatment outcomes with antidepressants
PMCID: PMC4227651  PMID: 25386878
24.  Lupus-prone New Zealand Black/New Zealand White F1 mice display endothelial dysfunction and abnormal phenotype and function of endothelial progenitor cells 
Lupus  2010;19(3):288-299.
Patients with systemic lupus erythematosus (SLE) have an impairment in phenotype and function of endothelial progenitor cells (EPCs) which is mediated by interferon α (IFN-α). We assessed whether murine lupus models also exhibit vasculogenesis abnormalities and their potential association with endothelial dysfunction. Phenotype and function of EPCs and type I IFN gene signatures in EPC compartments were assessed in female New Zealand Black/New Zealand White F1 (NZB/W), B6.MRL-Faslpr/J (B6/lpr) and control mice. Thoracic aorta endothelial and smooth muscle function were measured in response to acetylcholine or sodium nitropruside, respectively. NZB/W mice displayed reduced numbers, increased apoptosis and impaired function of EPCs. These abnormalities correlated with significant decreases in endolthelium-dependent vasomotor responses and with increased type I IFN signature in EPC compartments. In contrast, B6/lpr mice showed improvement in endothelium-dependent and endothelium-independent responses, no abnormalities in EPC phenotype or function and downregulation of type I IFN signatures in EPC compartments. These results indicate that NZB/W mice represent a good model to study the mechanisms leading to endothelial dysfunction and abnormal vasculogenesis in lupus. These results further support the hypothesis that type I IFNs may play an important role in premature vascular damage and, potentially, atherosclerosis development in SLE.
PMCID: PMC3151666  PMID: 20068018
endothelial progenitor cells; endothelium; interferon-alpha; systemic lupus erythematosus
25.  Analysis of T cell function in autoimmune murine strains. Defects in production and responsiveness to interleukin 2 
In the studies reported here, we have analyzed the production and consumption of T cell growth factor, more recently termed interleukin 2 (IL-2), as well as some cell-mediated immune functions, in murine strains [MRL, BXSB, NZB, and (NZB x NZWF1] manifesting systemic lupus erythematosus (SLE)-like syndromes. Young (4-6 wk) or old (4-8 mo) autoimmune or normal mice were studied and compared with regard to the following T cell functions in vitro after stimulation with concanavalin A (Con A): (a) mitogenic response; (b) IL-2 levels in culture supernates; and (c) the ability to respond to and adsorb IL-2. In addition, proliferative activity in the allogeneic mixed leukocyte culture and frequency of alloreactive cytotoxic T lymphocyte precursors (CTLp) were analyzed in some of these strains. Reduced Con A-induced mitogenic responses and IL-2 production appeared at 3-6 wk of age in the early, severe SLE developing strains MRL-Mp-lpr/lpr (MRL/l) and male BXSB and progressed thereafter. Similar defects appeared at a later stage in MRL/Mp-+/+ and (NZB x NZW)F1 hybrid mice, which develop late disease. Detailed analysis of cells from the enlarged lymph nodes and spleens of older MRL/l mice demonstrated that such cells: (a) responded poorly to Con A or allogeneic stimulator cells, even in the presence of exogenous IL-2; (b) did not suppress IL-2 production by normal spleen cells; (c) were relatively incapable of adsorbing or inactivating IL-2; and (d) had a markedly reduced anti-H-2b CTLp frequency in the mesenteric lymph nodes but a normal one in spleen. These results indicate that the proliferating Thy-1.2+, Lyt-1+ T cells in MRL/l mice are defective in their responses to mitogenic stimuli, in IL-2 production, and in expression of acceptor sites for IL-2. The relevance of these defects to the MRL/l disease as well as to the role of IL-2 in autoimmunity in general remains to be determined.
PMCID: PMC2186441  PMID: 6456321

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