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Background Recent studies have shown that early growth response 2 (EGR2) is highly induced in activated T cells and regulates T cell functions. In normal C57BL/6 (B6) mice, deletion of EGR2 in lymphocytes results in the development of lupus-like systemic autoimmune disease, which implies indirectly an autoimmune protective role of EGR2. Conversely, increased EGR2 gene expression is suggested to link with high risk of human lupus. In the present studies we sought to clarify the expression and inflammation regulatory role of EGR2 in murine lupus T cells directly. Results We performed RT-qPCR analysis and found a significant increase of EGR2 mRNA expression in human lupus PBMCs and in CD4+ T cells from three different murine lupus models including MRL-lpr, B6-lpr, and B6.sle123 mice at diseased stage when compared to age-matched control MRL or B6 mice. By performing intracellular flow cytometry analysis, we found that EGR2 protein expression was significantly increased in resting lupus (either MRL-lpr or B6.sle123) CD4+ T cells when compared to CD4+ T cells from their respective non-autoimmune controls. However, there was no difference of EGR2 protein expression in anti-CD3 and anti-CD28 stimulated control and lupus CD4+ T cells since there was a stronger induction of EGR2 in activated control CD4+ T cells. EGR2 expression was significantly increased in MRL-lpr mice at an age when lupus is manifested. To understand further the function of elevated EGR2 in lupus CD4+ T cells, we inhibited EGR2 with a specific siRNA in vitro in splenocytes from MRL-lpr and control MRL mice at 15 weeks-of-age. We found that EGR2 inhibition significantly reduced IFNγ production in PMA and ionomycin activated MRL-lpr lupus CD4+ T cells, but not control MRL CD4+ T cells. We also found that inhibition of EGR2 in vitro suppressed the Th1 differentiation in both MRL and MRL-lpr naïve CD4+ T cells. Conclusions EGR2 is highly upregulated in human and murine lupus cells. Our in vitro data suggest a positive role of EGR2 in the regulation of Th1 differentiation and IFNγ production in lupus effector CD4+ T cells.

MRL/lpr mice have been extensively used as a murine model of lupus. Disease progression in MRL/lpr mice can differ among animal facilities, suggesting a role for environmental factors.We noted a phenotypic drift of our in-house colony, which was the progeny of mice obtained from The Jackson Laboratory (JAX; stocking number 000485), that involved attenuated glomerulonephritis, increased splenomegaly, and reduced lymphadenopathy. To validate our in-house mice as a model of lupus, we compared these mice with those newly obtained from JAX, which were confirmed to be genetically identical to our in-house mice. Surprisingly, the new JAX mice exhibited a similar phenotypic drift, most notably the attenuation of glomerulonephritis. Interestingly, our in-house colony differed from JAX mice in body weight and kidney size (both sexes), as well as in splenic size, germinal center formation, and level of anti-dsDNA auto-IgG in the circulation (male only). In addition, we noted differential expression of microRNA (miR)-21 and miR-183 that might explain the splenic differences in males. Furthermore, the composition of gut microbiota was different between in-house and new JAX mice at early time points, which might explain some of the renal differences (e.g., kidney size). However, we could not identify the reason for attenuated glomerulonephritis, a shared phenotypic drift between the two colonies. It is likely that this was due to certain changes of environmental factors present in both JAX and our facilities. Taken together, these results suggest a significant phenotypic drift in MRL/lpr mice in both colonies that may require strain recovery from cryopreservation. This work was supported by internal funding from Virginia-Maryland College of Veterinary Medicine and National Institutes of Health Grants AR067418 and AR073240 (X.M.L.). Published version

Two variants of extracellular β-glucosidase (BGL2) were purified from the stipe and pilei of

ChiEn3 from Coprinopsis cinerea was characterized as an exo-acting chitinase with a processivity. ChiEn3 hydrolyzed only soluble chitin and exhibited a hyperhydrolytic activity toward 85% deacetylated chitosan which was 33.6-fold higher than its hydrolytic activity toward glycol chitin. Its maximum hydrolytic activity was observed at 60 °C and retained 66.2% of hydrolytic activity after 60 min incubation at 60 °C. Commercial 85% deacetylated chitosan was degraded by ChiEn3 to a series of COSs with a DP of 2–20 in which COSs with a DP of 3–6 were dominant, whereas, lab-prepared chitosan (FA = 0.65) was degraded by ChiEn3 to COSs with a DP of 2–10 in which the AA dimer was dominant. DPPH-radical-scavenging activity of ChiEn3-digested products of 85% deacetylated chitosan was 3.32-fold higher than that of undigested 85% deacetylated chitosan. Therefore, ChiEn3 shows a valuable advantage for application to the preparation of COSs from commercial 85% deacetylated chitosan.

Transcription factor early growth response protein 2 (EGR2) prevents the development of lupus-like autoimmune disease in C57BL/6 (B6) mice by negatively regulating T cell activation, inflammatory cytokine IFNγ and IL-17 production and by suppressing humoral responses. However, increased EGR2 expression has been observed in lupus and also other autoimmune disorders, which contradicts the autoimmune suppressive role of EGR2. Here, we derived conditional EGR2−/−B6/lpr and EGR2−/−B6 mice to investigate and compare the immune regulatory role of EGR2 in autoinflammation (B6/lpr) versus normal physiological (B6) conditions. Significantly, we found that in B6/lpr mice, but not in B6 mice, EGR2 deletion dramatically suppressed serum levels of anti-dsDNA autoantibodies, total IgG, IgG1, IgG2a, and IgM. We further demonstrated that while EGR2 deletion promoted germinal center B (GCB) cell development in both B6 and B6/lpr mice, it significantly reduced the differentiation of plasma and plasmablasts cells in B6/lpr mice. EGR2 deletion promoted splenomegaly and T cell activation in both B6 and B6/lpr mice, but it differentially regulated the profiles of immune cell subpopulations in the spleens of B6 and B6/lpr mice. Especially, EGR2 deletion dramatically suppressed the development of double negative T cells in B6/lpr mice. Moreover, we found that EGR2 had an opposite role in regulating IL-17 production in in vitro activated splenocytes from B6/lpr and B6 mice, although EGR2 had a similar suppressive role for IFNγ production. Together, our data strongly suggest that the immune and autoimmune regulatory roles of EGR2 are context dependent, and should not be generalized in normal physiology and different pathological conditions. Supported by VMCVM-VCOM Center for One Heath Research Seed Grant Program

Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease. The cause of SLE is not only genetic; in fact, environmental factors may play a more important role in disease development. The MRL/lpr mouse model lupus-like symptoms due to multiple SLE susceptible loci in the MRL background, and offers an accelerated model compared to the MRL parent strain due to the Faslpr mutation. Recently, our laboratory witnessed a loss of disease phenotype in our in-house colony of MRL/lpr mice. We thus compared mice newly obtained from The Jackson Laboratory (JAX; Stock Number 000485) to our in-house mice. The single-nucleotide polymorphism (SNP) analysis showed no genetic drift, suggesting that environmental factors could be triggering a phenotypic drift. Surprisingly, the newly purchased JAX mice also had attenuation of glomerulonephritis. Even though JAX mice manifested similar attenuation of lupus nephritis, our in-house colony showed differences in organ weights. Furthermore, males showed a significantly higher level of anti-double stranded DNA auto-IgG consistent with germinal center maturation. In addition, in-house males had significantly higher levels of microRNA-21 and microRNA-183 explaining spleen size difference. Moreover, the composition of gut microbiota was different between in-house and new JAX mice at early age, with many groups of bacteria differing at later time points, which might explain some of the phenotypic differences. These results suggest that microRNAs and gut microbiota might be responsible for the phenotypic differences of MRL/lpr mice in JAX and our colonies as they were genetically identical. On the other hand, the attenuation of nephritis in both groups requires further investigation. Supported by R01AR073240

Estrogens have been shown to regulate the immune system and modulate multiple autoimmune diseases. 17α-ethinyl estradiol (EE), a synthetic analog of 17β-estradiol, is prescribed commonly and found in oral contraceptives and hormone replacement therapies. Surprisingly, few studies have investigated the immunoregulatory effects of exposure to EE, especially in autoimmunity. In this study, we exposed autoimmune-prone female MRL/lpr mice to a human-relevant dose of EE through the oral route of exposure. Since lupus patients are prone to infections, groups of mice were injected with viral (Imiquimod, a TLR7 agonist) or bacterial (ODN 2395, a TLR9 agonist) surrogates. We then evaluated autoimmune disease parameters, kidney disease, and response to in vivo TLR7/9 pathogenic signals. EE-exposed mice had increased proteinuria as early as 7 weeks of age. Proteinuria, blood urea nitrogen, and glomerular immune complex deposition were also exacerbated when compared to controls. Production of cytokines by splenic leukocytes were altered in EE-exposed mice. Our study shows that oral exposure to EE, even at a very low dose, can exacerbate azotemia, increase clinical markers of renal disease, enhance glomerular immune complex deposition, and modulate TLR7/9 cytokine production in female MRL/lpr mice. This study may have implications for EE-exposure risk for genetically lupus-prone individuals. Preparation of this publication was supported by the Virginia-Maryland College of Veterinary Medicine (VMCVM) Intramural Research Competition (IRC) Grant (grant number 175185); AH&D USDA-NIFA grant (1016123), Interdepartmental funds to SAA, and by the National Institute of Health T32 training grant (grant number 5T32OD010430-09). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or VMCVM. We would like to acknowledge Virginia Tech’s OASF financial support for publication.

17α-Ethinyl estradiol (EE), a synthetic analog of natural estrogen 17β-estradiol (E2), is extensively used in hormonal contraceptives and estrogen replacement therapy, and it has also been found in sewage effluents. Given that E2 is a well-known immunomodulator, surprisingly there has been only limited information on the cellular and molecular immunologic consequences of exposure to EE. To address this fundamental gap, we directly compared the effects of EE with E2 on splenic leukocytes of New Zealand Black × New Zealand White F(1) progeny (NZB/W(F1)) mice during the preautoimmune period. We found that EE and E2 have common, as well as distinctive, immunologic effects, with EE exposure resulting in more profound effects. Both EE and E2 increased numbers of splenic neutrophils, enhanced neutrophil serine proteases and myeloperoxidase expression, promoted the production of nitric oxide and monocyte chemoattractant protein-1, and altered adaptive immune T cell subsets. However, activation of splenic leukocytes through the T cell receptor or Toll-like receptor (TLR)4 revealed not only common (IL-10), but also hormone-specific alterations of cytokines (IFNγ, IL-1β, ΤΝFα, IL-2). Furthermore, in EE-exposed mice, TLR9 stimulation suppressed IFNα, in contrast to increased IFNα from E2-exposed mice. EE and E2 regulated common and hormone-specific expression of immune-related genes. Furthermore, EE exposure resulted in more marked alterations in miRNA expression levels than for E2. Only EE was able to reduce global DNA methylation significantly in splenic leukocytes. Taken together, our novel data revealed that EE and E2 exposure confers more similar effects in innate immune system–related cell development and responses, but has more differential regulatory effects in adaptive immune–related cell development and responses.

Although age-at-injury influences chronic recovery from traumatic brain injury (TBI), the differential effects of age on early outcome remain understudied. Using a male murine model of moderate contusion injury, we investigated the underlying mechanism(s) regulating the distinct response between juvenile and adult TBI. We demonstrate similar biomechanical and physical properties of naive juvenile and adult brains. However, following controlled cortical impact (CCI), juvenile mice displayed reduced cortical lesion formation, cell death, and behavioral deficits at 4 and 14 d. Analysis of high-resolution laser Doppler imaging showed a similar loss of cerebral blood flow (CBF) in the ipsilateral cortex at 3 and 24 h post-CCI, whereas juvenile mice showed enhanced subsequent restoration at 2–4 d compared with adults. These findings correlated with reduced blood–brain barrier (BBB) disruption and increased perilesional vessel density. To address whether an age-dependent endothelial cell (EC) response affects vessel stability and tissue outcome, we magnetically isolated CD31+ECs from sham and injured cortices and evaluated mRNA expression. Interestingly, we found increased transcripts for BBB stability-related genes and reduced expression of BBB-disrupting genes in juveniles compared with adults. These differences were concomitant with significant changes in miRNA-21-5p and miR-148a levels. Accompanying these findings was robust GFAP immunoreactivity, which was not resolved by day 35. Importantly, pharmacological inhibition of EC-specific Tie2 signaling abolished the juvenile protective effects. These findings shed new mechanistic light on the divergent effects that age plays on acute TBI outcome that are both spatial and temporal dependent.SIGNIFICANCE STATEMENTAlthough a clear “window of susceptibility” exists in the developing brain that could deter typical developmental trajectories if exposed to trauma, a number of preclinical models have demonstrated evidence of early recovery in younger patients. Our findings further demonstrate acute neuroprotection and improved restoration of cerebral blood flow in juvenile mice subjected to cortical contusion injury compared with adults. We also demonstrate a novel role for endothelial cell-specific Tie2 signaling in this age-related response, which is known to promote barrier stability, is heightened in the injured juvenile vasculature, and may be exploited for therapeutic interventions across the age spectrum following traumatic brain injury.

The miR-183-96-182 (miR-183C) is a highly conserved miRNA cluster among species. Our previous work found a significant upregulation of miR-183C in the splenic cells of three different murine models of systemic lupus erythematosus (SLE). Current studies revealed that miR-183C miRNAs are critically involved in immunity and autoimmunity. In this study, we found that inhibition of miR-182 alone or miR-183C in vitro with antagomirs significantly reduced lupus-related inflammatory cytokine IFN-γ and IL-6 in activated splenocytes from MRL or MRL/lpr mice. To further characterize the pathogenic role of miR-182 and miR-183C in lupus in vivo, we developed B6-lpr mice with conditional depletion of miR-182 or miR-183C in CD2+ lymphocyte. We found that depletion of either miR-182 or miR-183C in the lymphocytes of B6/lpr mice had no obvious effect on T and B cell development as similar percentage of CD4+. CD8+, CD19+, as well as Tregs, follicular helper T (TFH), germinal center B (GCB), and plasma cells were observed in the miR-182−/− and miR-183C−/− and their respective control. Importantly, we observed a significant reduction of serum anti-dsDNA autoantibodies in miR-183C−/− mice when compared to age-matched controls and the B6/lpr mice with miR-182 or miR-183C deficiency have significantly reduced IgG deposition in the kidneys. Meanwhile, there was reduced IFN production in ex vivo activated splenocytes from the knockout mice. Furthermore, we demonstrated that miR-182 and miR-183C regulated the inflammatory response in splenocytes via targeting forkhead box O1 (Foxo1). Together, our data suggest a potential therapeutic effect of targeting miR-183C in lupus.

Early growth response protein 2 (EGR2) is a zinc-finger transcription factor that belongs to the early growth response (Egr) gene family. Recent studies have shown that EGR2 is required to induce T cell anergy, and it plays an important role in T cell immunity and autoimmunity. Conditional depletion of EGR2 in T lymphocytes in C57BL/6 (B6) mice (EGR2−/−B6) was able to break immune tolerance and induce T cell-driven, a lupus-like autoimmune disease. In EGR2−/−B6 mice, there was also enhanced GC response and humoral responses. However, increased EGR2 expression has been observed in lupus and autoimmune disorder systemic sclerosis. These observations seem to contradict the autoimmune suppressive role of EGR2 in B6 mice. To investigate further the immune regulatory role of EGR2 in an autoimmune context, we derived EGR2−/−B6/lpr mice with EGR2 deficiency in both T and B cells. We found that EGR2 depletion promoted splenomegaly and increased IFNγ, IL-10, IL-2 in both B6 and B6/lpr mice. Significantly, we found that in B6/lpr mice, EGR2 depletion reduced serum levels of anti-dsDNA autoantibodies, total IgG, IgG1, IgG2a, and IgM. Furthermore, in B6/lpr mice, depletion of EGR2 promoted germinal center B (GCB) cell development, but suppressed plasma cell (PC) differentiation. However, in wild-type B6 mice, EGR2 depletion significantly increased anti-dsDNA autoantibodies, accompanied by a significant increase of GCB cell development and a trend of increase in PC differentiation. Together, we demonstrated that EGR2 has a differential role in the normal and autoimmune state, particularly in regulating GCB transition into plasma cell during late B cell development.

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease that afflicts multiple organs, especially kidneys and joints. In addition to genetic predisposition, it is now evident that DNA methylation and microRNAs (miRNAs), the two major epigenetic modifications, are critically involved in the pathogenesis of SLE. DNA methylation regulates promoter accessibility and gene expression at the transcriptional level by adding a methyl group to 5′ cytosine within a CpG dinucleotide. Extensive evidence now supports the importance of DNA hypomethylation in SLE etiology. miRNAs are small, non-protein coding RNAs that play a critical role in the regulation of genome expression. Various studies have identified the signature lupus-related miRNAs and their functional contribution to lupus incidence and progression. In this review, the mutual interaction between DNA methylation and miRNAs regulation in SLE is discussed. Some lupus-associated miRNAs regulate DNA methylation status by targeting the DNA methylation enzymes or methylation pathway-related proteins. On the other hand, DNA hyper- and hypo-methylation are linked with dysregulated miRNAs expression in lupus. Further, we specifically discuss the genetic imprinting Dlk1-Dio3 miRNAs that are subjected to DNA methylation regulation and are dysregulated in several autoimmune diseases, including SLE.

Coprinopsis polysaccharides exhibit hypoglycemic and antioxidant activities. In this report, increases in polysaccharide production by homologous co-overexpression or individual homologous overexpression of phosphoglucomutase and UDP glucose pyrophosphorylase gene in Coprinopsis cinerea, which participate in polysaccharide biosynthesis. The transcription levels of the target genes were upregulated significantly in the oePGM-UGP strain when compared with the oePGM or oeUGP strain. The maximum intracellular polysaccharide content obtained in the oePGM-UGP strain was 1.49-fold higher than that of the WT strain, whereas a slight improvement in polysaccharide production was obtained in the oePGM and oeUGP strains. Extracellular polysaccharide production was enhanced by 75% in the oePGM-UGP strain when compared with that of the WT strain, whereas improvements of 30% and 16% were observed for the oePGM and oeUGP strains, respectively. These results show that multiple interventions in polysaccharide biosynthesis pathways of Basidiomycetes might improve polysaccharide yields when compared with that of single interventions.

Consequential differences exist between the male and female immune systems’ ability to respond to pathogens, environmental insults or self-antigens, and subsequent effects on immunoregulation. In general, females when compared with their male counterparts, respond to pathogenic stimuli and vaccines more robustly, with heightened production of antibodies, pro-inflammatory cytokines, and chemokines. While the precise reasons for sex differences in immune response to different stimuli are not yet well understood, females are more resistant to infectious diseases and much more likely to develop autoimmune diseases. Intrinsic (i.e., sex hormones, sex chromosomes, etc.) and extrinsic (microbiome composition, external triggers, and immune modulators) factors appear to impact the overall outcome of immune responses between sexes. Evidence suggests that interactions between environmental contaminants [e.g., endocrine disrupting chemicals (EDCs)] and host leukocytes affect the ability of the immune system to mount a response to exogenous and endogenous insults, and/or return to normal activity following clearance of the threat. Inherently, males and females have differential immune response to external triggers. In this review, we describe how environmental chemicals, including EDCs, may have sex differential influence on the outcome of immune responses through alterations in epigenetic status (such as modulation of microRNA expression, gene methylation, or histone modification status), direct and indirect activation of the estrogen receptors to drive hormonal effects, and differential modulation of microbial sensing and composition of host microbiota. Taken together, an intriguing question develops as to how an individual’s environment directly and indirectly contributes to an altered immune response, dysregulation of autoantibody production, and influence autoimmune disease development. Few studies exist utilizing well-controlled cohorts of both sexes to explore the sex differences in response to EDC exposure and the effects on autoimmune disease development. Translational studies incorporating multiple environmental factors in animal models of autoimmune disease are necessary to determine the interrelationships that occur between potential etiopathological factors. The presence or absence of autoantibodies is not a reliable predictor of disease. Therefore, future studies should incorporate all the susceptibility/influencing factors, coupled with individual genomics, epigenomics, and proteomics, to develop a model that better predicts, diagnoses, and treats autoimmune diseases in a personalized-medicine fashion. Virginia- Maryland College of Veterinary Medicine VMCVM: 175185 National Institutes of Health NIH: 5T32OD010430-09

Unique dysregulated expression patterns of microRNAs (miRNAs) have been reported in many disease conditions including autoimmune diseases such as systemic lupus erythematosus (SLE). We have recently reported that miRNAs are differentially expressed not only between autoimmune and control mice, but also between male and female lupus-prone mice. This important observation is of potential clinical and experimental significance since females have higher incidence, earlier expression or severity of lupus when compared to their male counterparts. Further, estrogen administration to orchiectomized males accelerates the expression of lupus-related miRNAs and induces unique miRNA signature profile. Understanding the basis of altered miRNAs in autoimmune diseases offers a new paradigm to understand autoimmune diseases, including sex-differential susceptibility. In this review, we summarize miRNA biogenesis and function, and focus on miRNA dysregulation in SLE in the context of sexual bias. Furthermore, the effect of estrogen on epigenetic miRNA regulation in relation to SLE is highlighted.

To resolve the structure of laminarin, the recombinant endo-β-1,3-glucanase from Coprinopsis cinerea, which has specific activity on β-1,3 glycosidic bond and could hydrolyze the laminarin with complex structure, was used to hydrolyze laminarin. Then, the structures of enzyme-resistant oligosaccharides were quantitatively and qualitatively analysed by high-performance anion exchange chromatography coupled with mass spectrometry. The laminarin from Laminaria digitata contains 9.51% β-1,6 glycosidic bonds only in the branches (branch degree 7.68%). The laminarin from Eisenia bicyclis contains more β-1,6 glycosidic bonds: 19.42% β-1,6 glycosidic bonds in backbone and more and longer β-1,6 branches (branch degree 25.99%). The differences in the ratio of glycosidic bonds and branch degree influence their bioactivity: the antioxidant activity and the antimicrobial activity against Gram positive bacteria of the laminarin from E. bicyclis is stronger than the laminarin from L. digitata, but the antimicrobial activity on Gram negative bacteria of the laminarin from E. bicyclis is weaker.

Background and aims Oestrogen, a natural immunomodulator, regulates the development and function of diverse immune cell types and has been implicated in lupus development. Methods To determine the regulatory role of oestrogen on neutrophil development and function, we treated B6 mice with placebo- or oestrogen implants for 6–8 weeks, and then analysed splenic neutrophil serine proteases (NSPs, such as neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (CG)) and myeloperoxidase (MPO) expression by qRT-PCR. NE -/- /PR3 -/- /CG -/- triple knockout mice and in vitro depletion of neutrophils approaches were performed to determine the role of NSPs and neutrophils in estrogen-mediated inflammatory responses. The splenic neutrophil and NSPs expression in lupus-prone MRL- lpr , B6- lpr and NZB/W F1 and their respective controls were also analysed. Results Although oestrogen reduced total splenocytes number, it markedly increased the splenic neutrophil numbers, NSPs and MPO expression in B6 mice (Figure 1). Splenic neutrophils, NSPs and MPO were also significantly increased in MRL- lpr, B6- lpr and NZB/W F1 mice (Figure 2). Despite of the critical role of NSPs and neutrophils in inflammation, depletion of NSPs in vivo did neither affect oestrogen’s ability to increase in splenic neutrophils nor the induction of inflammatory mediators from ex vivo activated splenocytes, and depletion of splenic neutrophils in vitro had also no obvious effect on NSPs expression (due to the increase of NSPs in cells other than neutrophils) and LPS-induced IFNg and MCP-1 (Figure 3). Conclusions Overall, we demonstrated a remarkable commonality with regards to the increase of neutrophils and NSPs in the spleens of autoimmune-prone mice and estrogen-treated B6 mice.

To date, there are 18 histone deacetylase (HDAC) enzymes, divided into four classes, which alter protein function by removing acetyl groups from lysine residues. Prior studies report that non-selective HDAC inhibitors decrease disease in lupus mouse models. Concern for adverse side effects of non-selective HDAC inhibition supports investigation of selective-HDAC inhibition. We hypothesized that a selective HDAC-6 inhibitor (HDAC6i) will alleviate disease in a mouse model of lupus by increasing acetylation of alpha-tubulin. Intraperitoneal injections of the selective HDAC6i ACY-1083 (0.3 mg/kg, 1 mg/kg, or 3 mg/kg), vehicle control, or dexamethasone were administered to 21-week-old, female NZB/W mice, 5 days a week, for 13 weeks. Disease progression was evaluated by proteinuria, serum levels of anti-dsDNA antibody, cytokines and immunoglobulins, and post mortem evaluation of nephritis and T cell populations in the spleen. HDAC6i treatment decreased proteinuria, glomerular histopathology, IgG, and C3 scores when compared to vehicle-treated mice. Within glomeruli of HDAC6i-treated mice, there was increased acetylation of alpha-tubulin and decreased NF-κB. Additionally, HDAC6i decreased serum IL-12/IL-23 and Th17 cells in the spleen. Taken together, these results suggest HDAC-6 inhibition may decrease lupus nephritis in NZB/W mice via mechanisms involving acetylation of alpha-tubulin and decreased NF-κB in glomeruli as well as inhibition of Th17 cells.

MicroRNAs (miRNAs), although small in size, have attracted the attention of cell biologists and clinicians alike for their ability to regulate genome expression at the posttranscriptional level. Within two decades, miRNAs have surged to the forefront as key epigenetic regulators of diverse biological systems including the immune system in both physiological processes and pathological situations. This chapter updates our recent review article (Dai R. & Ahmed S. Ansar, Translational Research 2011; 157:163–170) with current understanding of miRNA biogenesis, miRNA regulation of innate and adaptive immunity in physiological conditions, and miRNA dysregulation in pathological states with a focus on a prototypical autoimmune disease systemic lupus erythematosus (SLE). The dysregulated miRNAs in human and murine lupus have been reported to contribute to lupus pathogenesis by regulating DNA methylation, T- and B-cell development and function, and the induction of lupus-related inflammatory cytokines and chemokines. The miRNA dysregulation during lupus pathogenesis may be caused by multiple factors comprising genetic, epigenetic, hormonal, and environmental factors. Given that SLE and many other autoimmune diseases predominantly affect females, we specifically discuss the female hormone estrogen regulation of miRNA and the sexual dimorphism of miRNA expression in the SLE setting. The stable identification of unique, signature cellular, and noncellular miRNA expression profiles that correlate with the onset/severity of autoimmune disease such as SLE holds promise as novel, and much needed diagnostic biomarkers for autoimmune disease. With a comprehensive understanding of the pathogenic role of miRNA in autoimmune disease and advent of innovative techniques for effective targeting of miRNA in vivo, we anticipate the development of miRNA-based therapeutic strategies in the near future for treating SLE and other inflammatory autoimmune diseases in clinical applications.

We have previously reported a significant upregulation of microRNA-183-96-182 cluster (miR-183C) in splenocytes of several murine lupus models. In this study, we found that the upregulation of miR-183C is inversely related to the expression of Foxo1 and Foxo3a, which play important roles in the regulation of T lymphocyte homeostasis and immune responses. Importantly, we found that inhibition of miR-183C with specific antagomirs significantly reduces lupus-related inflammatory cytokines expression such as IFNg, IL-6, and IL-17 in in vitro activated splenocytes from both MRL and MRL-lpr mice. We further showed that inhibition of miR-183C increases expression of Foxo1 and that inhibition of Foxo1 with siRNA increases IFNg and IL-6 in activated splenocytes from MRL mice. These data suggest that the miR-183C may regulate inflammatory response by targeting Foxo1. To further characterize the role of miR-183C in lupus pathogenesis, we developed B6-lpr mice with conditional depletion of miR-183C in T and B lymphocyte (miR-183C−/− B6-lpr) by serial crossbreeding of B6-lpr with miR-183Cfl/fl and hCD2-iCre mice. To our surprise, there was no significant difference in the production of IFNg and IL-6 in in vitro activated splenocytes from knock out (miR-183C−/− B6-lpr) mice when compared to that from control (miR-183Cfl/fl B6-lpr) mice. Impressively, we found that miR-183C−/− B6-lpr had reduced serum anti-dsDNA levels, spleen and lymphoid node weight when compared to controls. The histopathological analysis also revealed reduced inflammation in kidney in miR-183C−/− B6-lpr mice. Together, our results suggest a potential role of miR-183C in lupus pathogenesis and that miR-183C might be a new potential target for lupus therapy.

Recent evidence supports a role for epigenetic alterations in the pathogenesis of systemic lupus erythematosus (SLE). MicroRNAs (miRNAs or miRs) are endogenous epigenetic regulators whose expression is altered in many diseases, including SLE. IL-6 is an inflammatory cytokine produced by mesangial cells during lupus nephritis (LN). IL-6 contains a potential binding site for miRNA-let-7a (let-7a) in its 3′ untranslated region (UTR). We found let-7a expression was significantly increased in the mesangial cells of pre-diseased and actively diseased New Zealand Black/White (NZB/W) mice compared to age-matched New Zealand White (NZW) mice. Overexpression of let-7a in vitro increased IL-6 production in stimulated mesangial cells compared to non-transfected controls. Inhibition of let-7a did not significantly affect immune-stimulated IL-6 production. When stimulated mesangial cells overexpressing let-7a were treated with the transcription inhibitor Actinomycin D (ActD), IL-6 was degraded faster, consistent with the direct targeting of the 3′ UTR of IL-6 by let-7a. Overexpression of let-7a increased the expression of tristetraprolin (TTP), an RNA-binding protein (RBP) that has 5 potential binding regions in the 3′ UTR of IL-6. ActD inhibited the transcription of proteins including TTP that may contribute to the let-7a-mediated increase in immune-stimulated IL-6 production. These data show that NZB/W mice have higher let-7a expression than NZW mice and that increased let-7a expression in vitro increases IL-6 production in stimulated mesangial cells. Further studies examining the role of let-7a expression in inflammation are warranted.

The course and severity of lupus in spontaneous murine lupus models varies among laboratories, which may be due to variations in diet, housing and/or local environmental conditions. In this study, we investigated the influence of common rodent diets while keeping other factors constant. Female lupus-prone MRL/lpr (MRL/MpJ-Faslpr/J) mice were subjected to the same housing conditions and given one of the three diets: Teklad 7013 containing isoflavone-rich soy and alfalfa, Harlan 2018 isoflavone-rich soy-based diet or Research Diets Inc. D11112226 (RD) purified-ingredients diet containing casein and no phytoestrogens. While the total caloric intake was similar among all three treatment groups, mice fed on the 2018 diet developed higher levels of proteinuria and mice fed on either 7013 or 2018 developed higher levels of glomerular immune complex deposition. Remarkably, mice fed the RD diet had markedly decreased proteinuria with diminished C3, total IgG, IgG1 and IgG3 immune complex deposition, along with reduced CD11b+ cellular infiltration into the glomeruli. The type of diet intake also influenced cytokine production, fecal microbiota (increased Lachnospiraceae in mice fed on 2018), altered microRNAs (miRNAs; higher levels of lupus-associated miR-148a and miR-183 in mice fed on 7013 and/or 2018) and altered DNA methylation. This is the first study to comprehensively compare the cellular, molecular and epigenetic effects of these commercial diets in murine lupus.

microRNAs (miRNAs) are newly discovered, small, non-coding ribonucleic acids (RNAs) that play critical roles in the regulation of host genome expression at the post-transcriptional level. During last two decades, miRNAs have emerged as key regulators of various biological processes including immune cell lineage commitment, differentiation, maturation, and maintenance of immune homeostasis and normal function. Thus, it is not surprising that dysregulated miRNA expression patterns have now been documented in a broad range of diseases including cancer, inflammatory, and autoimmune diseases. This rapidly emerging field has revolutionized our understanding of normal immunoregulation and breakdown of self-tolerance. This review focuses on the current understanding of miRNA biogenesis, the role of miRNAs in the regulation of innate and adaptive immunity, and the association of miRNAs with autoimmune diseases. We have discussed miRNA dysregulation and the potential role of miRNAs in systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and multiple sclerosis (MS). Given that most autoimmune diseases are female-predominant, we have also discussed sex hormone regulation of miRNAs in inflammatory responses, with an emphasis on estrogen, which has now been shown to regulate miRNAs in the immune system. The field of miRNA regulation of mammalian genes has tremendous potential. Identification of specific miRNA expression patterns in autoimmune diseases and further a comprehensive understanding of the role of miRNA in disease pathogenesis offers promise of not only novel molecular diagnostic markers, but also new gene therapy strategies for treating SLE and other inflammatory autoimmune diseases.

Estrogen-mediated regulation of Th1, Th2 and Treg effector functions are well documented but, surprisingly, there is little information whether estrogen modulates IL-17, a powerful proinflammatory cytokine that plays a pivotal role in several inflammatory and autoimmune diseases. Therefore in the current study, we determined whether estrogen regulates the expression levels of IL-17 in WT C57BL/6 mice. By ELISA, ELISPOT and/or flow cytometric analyses, we found that estrogen upregulated the levels of not only IL-17, but also the IL-17-specific transcription factor retinoic acid-related orphan receptor gamma t (ROR gamma t), in activated splenocytes. IL-17 levels were further enhanced by exposure of activated splenocytes to IL-23, particularly in cells from estrogen-treated mice. Exposure of splenocytes to IL-27 or IFN-gamma at the time of activation markedly inhibited the levels of IL-17 and ROR gamma t. Interestingly, a delay of 24 h in exposure of activated splenocytes to IL-27 or IFN-gamma decreased IL-17 levels (albeit less profoundly) but not ROR gamma t. These findings imply that the suppressive effects of IL-27 and IFN-gamma are more effective prior to the differentiation and commitment of IL-17-secreting cells. Furthermore, inhibition of JAK-2 by AG490 suppressed IL-17 but not ROR gamma t expression, suggesting that other transcription factors are also critical in estrogen-mediated upregulation of IL-17.

Concern is increasing regarding the effects of environmental exposures to different Estrogenic Endocrine Disrupting Chemicals (EEDCs) on immune system function. 17α-ethinyl estradiol (EE), a synthetic analog of natural estrogen 17-β estradiol (E2), is a primary component in oral contraceptive pills (OCP) and has been found in sewage effluents. In this study, to compare the immune and epigenetic regulatory effects of environmental E2 and EE exposure in NZB/WF1 mice, we implanted gonadal intact female NZB/WF1 mice with E2 or EE. While the effect of EE was more profound, both E2 and EE exposures caused splenomegaly, altered splenic and thymic T cell composition and activation, increased splenic neutrophil percentage, and promoted inflammatory molecules iNOS/NO and the chemokine MCP-1 in ex vivo-activated splenocytes. Impressively, we found that E2 and EE had differential effects on the induction of different inflammatory cytokines in ex vivo activated splenocytes (e.g. IFNβ and IL-1β were increased in LPS-activated splenocytes from EE-treated NZB/WF1 mice, but were decreased in LPS-activated splenocytes from E2-treated mice when compared to placebo controls). Also, in vivo EE exposure promoted, and E2 reduced, TLR9 agonist-induced IFNα production in splenocytes. We further demonstrated that E2 and EE regulated the expression of numerous immune-related genes and miRNAs in a similar manner, while EE had a stronger effect than E2 on the regulation of miRNAs expression. Moreover, we found that EE, but not E2 treatment, significantly reduced the global DNA methylation level in NZB/WF1 splenocytes. Together, our data revealed similar and also differential regulatory effects of E2 and EE on the different aspects of immune function.

Experimental autoimmune encephalomyelitis (EAE) is a CD4+ T-cell mediated disease, which resembles immunopathology of multiple sclerosis (MS). Interleukin (IL)-16 is a CD4+ cell-specific chemoattractant cytokine. In CD4+ T cells, production of bioactive IL-16 from constitutive pro-IL-16 requires cleavage by active caspase-3. We reported reversal of established relapsing disease by IL-16 neutralization. To better understand role(s) of IL-16 in regulation of relapsing EAE, we comparatively analyzed levels of IL-16, active caspase-3 and CD4 in mice with severe relapsing-remitting [(B6 × SJL) F1], and low-relapsing (B6), disease. Elevated levels of IL-16 along with an increase in active-caspase-3 and CD4 levels correlated with stages of clinically active disease in both strains. CNS levels of bioactive IL-16 were notably higher in F1 compared to B6 mice at all stages, being most prominent during relapse. Similar patterns of regulation for IL-16 and active caspase-3 were observed in peripheral lymphoid organs, and in T cells isolated from lymph nodes following T-cell activation in vitro. IL-16 was co-immunoprecipitated with CD4 from CNS of relapsing mice. Our data suggest that caspase-3 mediated production of IL-16 by infiltrating CD4+ T cells, contributes to ongoing neuroinflammation by chemoattraction of additional waves of CD4+ T cells.

A novel zebrafish gene bloody fingers (blf) encoding a 478 amino acid protein containing fifteen C2H2 type zinc fingers was identified by expression screening. As determined by in situ hybridization, blf RNA displays strong ubiquitous early zygotic expression, while during late gastrulation and early somitogenesis, blf expression becomes transiently restricted to the posterior dorsal and lateral mesoderm. During later somitogenesis, blf expression appears only in hematopoietic cells. It is completely eliminated in cloche, moonshine but not in vlad tepes (gata1) mutant embryos. Morpholino (MO) knockdown of the Blf protein results in the defects of morphogenetic movements. Blf-MO-injected embryos (morphants) display shortened and widened axial tissues due to defective convergent extension. Unlike other convergent extension mutants, blf morphants display a split neural tube, resulting in a phenotype similar to the human open neural tube defect spina bifida. In addition, dorsal ectodermal cells delaminate in blf morphants during late somitogenesis. We propose a model explaining the role of blf in convergent extension and neurulation. We conclude that blf plays an important role in regulating morphogenetic movements during gastrulation and neurulation while its role in hematopoiesis may be redundant.

Calcyon was recently identified as a D1 dopamine receptor (DR1) interacting protein. Previous studies show that calcyon can potentiate DR1 mediated intracellular Ca 2+ release in transfected HEK293 cells, and may play an important role in DR1 Ca 2+ signaling in brain. We report that similar to the genomic structure of the human gene, the mouse calcyon gene contains six relatively short exons, with a large intron (about 8.4 kb) between exons one and two. The mouse and human calcyon genes exhibit a high level of sequence homology (77.5% at the nucleotide level) within coding regions. Northern blot and RT-PCR analyses reveal that mouse calcyon transcripts are most abundant in brain, but also present in testis and ovary, as well as in kidney and heart at much lower levels. The most distal of the transcript initiation sites identified by 5′ RACE is located 159 nucleotides upstream of the putative start of translation. BLAST search of the NCBI mouse EST database and RT-PCR analysis uncovered two differentially spliced transcripts, ‘mcal-A’ and ‘mcal-B.’ The two transcripts are identical, except that mcal-B contains a longer 3′ untranslated region due to retention of a short intron (I5) between exons five and six. However, mcal-A represents the predominant calcyon transcript in mouse tissue. Further, the presence of I5 produced no detectable differences in the biosynthesis of calcyon polypeptide when expressed in HEK293, MDCK and Neuro2a cells.

The product of the obesity gene, called leptin, is an important regulator of adiposity, which mainly functions via its regulation of feed intake and energy metabolism. Both obesity gene (ob gene) and leptin receptor gene (OBR gene) are thought to play a major role in controlling of body fat mass as well as body weight. The result of RT-PCR shows that levels of pigOBR mRNA are higher in hypothalamus, lung and liver, and lower expression can be detected in other tissues. Total RNA purified from 11 different organs and tissues have been hybridized with pigOBR cDNA probes. The hybridization signals are shown in 7 organs and tissues. 4.1 and 3.8 kb bands were observed from hypothalamus, whereas 3.8 and 3.5 kb bands were observed in other tissues instead. The nearly complete sequence of the extracellular domain of pigOBR gene was obtained. The homology of sequence is 89.2% between pig and human, 80.3% between pig and mouse. Alignment of the predicted amino acid sequence ofOBR in pig, human and mouse shows that the overall identity is 86.5% between pig and human, 76.6% between pig and mouse. Two WSXWS motifs were found at aa313and aa6162.

Heat shock factor-1 (HSF-1) activates transcription of heat shock proteins in eukaryotes. Several overlapping genomic clones containing the murine HSF-1 gene were isolated from a phage genomic library. Results indicate that the HSF-1 gene contains 13 exons that span at least 30 kilobase pairs. Sequence analysis of the 5'-untranslated region of HSF-1 suggests that it contains sequences of a recently described Bop1 gene in reverse orientation within its first 331 base pairs (bp) upstream of the translation initiation site. The minimal promoter sequence required for HSF-1 basal expression was identified by deletion analysis from -4 kilobase pairs to -331 bp of the promoter fused to a luciferase reporter gene using transient transfection assays. Results indicate that 331 bp upstream of the HSF-1 translation start site is required for maximal basal expression in NIH3T3 and F9 cells. This fragment also results in high levels of luciferase activity in the reverse orientation, that is, 5' to the Bop1 gene, suggesting that this segment is bidirectional and could be utilized for basal expression of both HSF-1 and Bop1 genes. This segment of the promoter contains recognition elements for Sp1 and CCAAT-box binding transcription factors, which when mutated in either sense or antisense orientations to the HSF-1 gene results in a reduction of basal expression by 50-75% relative to wild type, suggesting that these sites are critical for basal expression of both HSF-1 and Bop1 genes.

Systemic lupus erythematosus is a prototypical autoimmune disease, characterized by the loss of immune cell tolerance to self-antigens, and the subsequent production of autoantibodies leading to multi-organ damage. SLE is an incurable, highly variable autoimmune disease that most often affects women of child-bearing age. We investigated the effects of three standard commercial diets with varying phytoestrogen content on the development of lupus in female MRL/lpr mice. The diets include Harlan 2018 which contains soybean meal, Teklad 7013 which contains both soy and alfalfa, and Research Diets Inc. D11112226 (RD) purified ingredients diet, containing no measurable phytoestrogens. Mice fed the 2018 diet developed severe glomerulonephritis with high levels of complement C3 protein deposition and moderate IgG immune-complex deposition, while mice fed the RD diet developed minimal evidence of renal damage and protein deposition. The mice fed the RD diet, however, had increased levels of multiple pro-inflammatory cytokines, while immune cell populations remained unchanged. Immunofluorescence revealed distinct infiltrating cellular phenotypes in the kidney, dependent on dietary source. Splenocyte miRNA-148a was markedly elevated in mice fed the 2018 and 7013 diets, while circulating miRNA values were not statistically different. Our results suggest that dietary isoflavones can worsen the development of glomerulonephritis due to enhanced immune complex protein deposition and altered cellular infiltration phenotypes. Our results also support that each organ system must be evaluated independently, and that what is measured in circulation does not necessarily reflect what is occurring in disease target organs.

Epigenetic factors such as DNA methylation and microRNAs (miRNAs) are now increasingly recognized as vital contributors to lupus etiology. In this study, we investigated the potential interaction of these two epigenetic factors in lupus-prone MRL-lpr mice. We recently reported dysregulated expression of miRNAs in splenocytes of MRL-lpr mice. Here, we report that a majority of the upregulated miRNAs in MRL-lpr mice is located at the genomic imprinted DLK1-Dio3 domain. Further, we show a differential magnitude of upregulation of DLK1-Dio3 miRNA cluster in purified splenic CD4+ T, CD19+ B, and splenic CD4-CD19- cells from MRL-lpr lupus mice when compared to control MRL mice. MRL-lpr splenocytes (especially CD19+ and CD4-CD19- subsets) were hypomethylated compared to cells from control, MRL mice. We further show that deliberate demethylation of splenocytes from control MRL mice, but not from MRL-lpr lupus mice, with specific DNA methylation inhibitor 5-Aza-2’-deoxycytidine significantly augmented DLK1-Dio3 miRNAs expression. These findings strongly indicate that the upregulation of DLK1-Dio3 miRNAs in lupus splenic cell subsets is associated with reduced global DNA methylation levels in lupus cells. There was a differential upregulation of DLK-Dio3 miRNAs among various demethylated splenic cell subsets, which implies varied sensitivity of DLK1-Dio3 miRNA cluster in these cell subsets to DNA hypomethylation. Finally, inhibition of select DLK1-Dio3 miRNA such as miR-154, miR-379 and miR-300 with specific antagomirs significantly reduced the production of lupus-relevant IFNγ, IL-1β, IL-6, and IL-10 in lipopolysaccharide (LPS) activated splenocytes from MRL-lpr mice. Our study is the first to show that DNA methylation regulates genomic imprinted DLK1-Dio3 miRNAs in autoimmune lupus, which suggests a connection of DNA methylation, miRNA and genomic imprinting in lupus pathogenesis.

Background 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. Conclusions/Significance 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.

Epigallocatechin-3-gallate (EGCG), a bioactive component of green tea, has been reported to exert anti-inflammatory effects on immune cells. EGCG is also shown to activate the metabolic regulator, adenosine 5'-monophosphate-activated protein kinase (AMPK). Reports have also indicated that EGCG inhibits the immune-stimulated phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway. The PI3K/Akt/mTOR pathway has been implicated in mesangial cell activation in lupus. Mesangial cells from MRL/lpr lupus-like mice are hyper-responsive to immune stimulation and overproduce nitric oxide (NO) and other inflammatory mediators when stimulated. In our current studies, we sought to determine the mechanism by which EGCG attenuates immune-induced expression of pro-inflammatory mediators. Cultured mesangial cells from MRL/lpr mice were pre-treated with various concentrations of EGCG and stimulated with lipopolysaccharide (LPS)/interferon (IFN)-gamma. EGCG activated AMPK and blocked LPS/IFN-gamma-induced inflammatory mediator production (iNOS expression, supernatant NO and interleukin-6). Interestingly, EGCG attenuated inflammation during AMPK inhibition indicating that the anti-inflammatory effect of EGCG may be partially independent of AMPK activation. Furthermore, we found that EGCG effectively inhibited the immune-stimulated PI3K/Akt/mTOR pathway independently of AMPK, by decreasing phosphorylation of Akt, suggesting an alternate mechanism for EGCG-mediated anti-inflammatory action in mesangial cells. Taken together, these studies show that EGCG attenuated inflammation in MRL/lpr mouse mesangial cells via the PI3K/Akt/mTOR pathway. Our findings suggest a potential therapeutic role for the use of EGCG to regulate inflammation and control autoimmune disease.

Estrogen regulation of inflammatory responses has broad physiological and pathological consequences. However, the molecular mechanism of estrogen regulation of inflammation is still poorly understood. In this study, we report that activation of both STAT-1 and NF-κB signaling is essential for Con A-induced inducible NO synthase (iNOS) and NO in murine splenocytes. Estrogen enhances STAT-1 DNA-binding activity without increasing the expression of phosphorylated and total STAT-1 protein. We have recently reported that estrogen blocks the nuclear expression of NF-κB p65 and modifies nuclear NF-κBp50. Here, we demonstrated that both nuclear STAT-1 and NF-κB are modified by serine protease-mediated proteolysis, which resulted in altered STAT-1 and NF-κB activity/signaling in splenocytes from estrogen-treated mice. Inhibition of serine protease activity with 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) restores the nuclear expression of full-length STAT-1 and NF-κB proteins, and resulted in decreased STAT-1 DNA-binding activity and formation of NF-κB p65/p50 binding complexes in nuclei of splenocytes from estrogen-treated mice. Consequently, there is significantly decreased iNOS and IFN-γ production in AEBSF-treated splenocytes from estrogen-treated mice, which suggests a positive regulatory role of truncated STAT-1 and/or NF-κB. Interestingly, there is increased production of MCP-1 in STAT-1 or NF-κB small interfering RNA-transfected cells, as well as in AEBSF-treated splenocytes from estrogen-treated mice. These data suggest a differential role of truncated STAT-1 and NF-κB in regulation of various inflammatory molecules in splenocytes from estrogen-treated mice. Together, our data reveal a novel molecular mechanism of estrogen-mediated promotion of inflammatory responses, which involves posttranslational modification of STAT-1 and NF-κB proteins.

Calcyon is a single transmembrane endocytic protein that regulates clathrin assembly and clathrin-mediated endocytosis in the brain. Ultrastructural studies indicate that calcyon localizes to spines, but whether it regulates glutamate neurotransmission is not known. Here, we show that deletion of the calcyon gene in mice inhibits agonist-stimulated endocytosis of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), without altering basal surface levels of the GluR1 or GluR2 subunits. Whole-cell patch-clamp studies of hippocampal neurons in culture and CA1 synapses in slices revealed that knockout (KO) of calcyon abolishes long-term synaptic depression (LTD), whereas mini-analysis in slices indicated basal transmission in the hippocampus is unaffected by the deletion. Further, transfection of green fluorescent protein-tagged calcyon rescued the ability of KO cultures to undergo LTD. In contrast, intracellular dialysis of a fusion protein containing the clathrin light-chain-binding domain of calcyon blocked the induction of LTD in wild-type hippocampal slices. Taken together, the present studies involving biochemical, immunological and electrophysiological analyses raise the possibility that calcyon plays a specialized role in regulating activity-dependent removal of synaptic AMPARs.

MicroRNAs (miRNAs), recently identified noncoding small RNAs, are emerging as key regulators in homeostasis of the immune system. Therefore, aberrant expression of miRNAs may be linked to immune dysfunction, such as in chronic inflammation and autoimmunity. In this study, we investigated the potential role of miRNAs in estrogen-mediated regulation of innate immune responses, as indicated by up-regulation of lipopolysaccharide (LPS)–induced interferon-gamma (IFNγ), inducible nitric oxide synthase (iNOS), and nitric oxide in splenic lymphocytes from estrogen-treated mice. We found that miR-146a, a negative regulator of Toll-like receptor (TLR) signaling, was decreased in freshly isolated splenic lymphocytes from estrogen-treated mice compared with placebo controls. Increasing the activity of miR-146a significantly inhibited LPS-induced IFNγ and iNOS expression in mouse splenic lymphocytes. Further, miRNA microarray and real-time reverse transcriptase–polymerase chain reaction (RT-PCR) analysis revealed that estrogen selectively up-regulates/down-regulates the expression of miRNAs in mouse splenic lymphocytes. miR-223, which is markedly enhanced by estrogen, regulates LPS-induced IFNγ, but not iNOS or nitric oxide in splenic lymphocytes. Inhibition of miR-223 activity decreased LPS-induced IFNγ in splenic lymphocytes from estrogen-treated mice. Our data are the first to demonstrate the selective regulation of miRNA expression in immune cells by estrogen and are indicative of an important role of miRNAs in estrogen-mediated immune regulation.

Estrogen, a natural immunomodulatory compound, has been shown to promote the induction of a prototype T helper 1 cytokine, interferon (IFN)-gamma, as well as to up-regulate IFN gamma-mediated pro-inflammatory molecules (nitric oxide, cyclooxygenase 2, monocyte chemoattractant protein 1). Because IL-12 is a major IFN gamma-inducing cytokine, in this study we investigated whether estrogen treatment of wild-type C57BL/6 mice alters IL-12-mediated signaling pathways. A recent study has shown that IL-12 activates two isoforms of signal transducer and activation of transcription (STAT) 4, a normal-sized (full-length STAT4 alpha) and a truncated form (STAT4 beta). Interestingly, we found that estrogen treatment preferentially up-regulates the phosphorylation of STAT4 beta in splenic lymphoid cells. Time kinetic data showed the differential activation of STAT4 beta in splenic lymphoid cells from estrogen-treated mice, but not in cells from placebo controls. The activation of STAT4 beta was mediated by IL-12 and not IFN gamma because deliberate addition or neutralization of IL-12, but not IFN gamma, affected the activation of STAT4 beta. In contrast to IL-12-induced activation of STAT4 beta in cells from estrogen-treated mice, STAT4 beta was not increased, rather it tended to be decreased. In this context, STAT4 alpha-induced p27(kip1) protein was decreased in concanavalin A + IL-12-activated lymphocytes from estrogen-treated mice only. By using the in vitro DNA binding assay, we confirmed the ability of pSTAT4 beta to bind to the IFN gamma-activated sites (IFN gamma activation sequences)/STAT4-binding sites in estrogen-treated mice. Our data are the first to show that estrogen apparently has selective effects on IL-12-mediated signaling by preferentially activating STAT4 beta. These novel findings are likely to provide new knowledge with regard to estrogen regulation of inflammation. (Endocrinology 150: 1310-1320, 2009) National Institutes of Health 5RO1 AI51880-03 United States Department of Agriculture-Hatch United States Department of Agriculture-Animal Health and Disease

D1-like dopamine receptors stimulate Ca(2+) transients in neurons but the effector coupling and signaling mechanisms underlying these responses have not been elucidated. Here we investigated potential mechanisms using both HEK 293 cells that stably express D1 receptors (D1HEK293) and hippocampal neurons in culture. In D1HEK293 cells, the full D1 receptor agonist SKF 81297 evoked a robust dose-dependent increase in Ca(2+)(i) following 'priming' of endogenous G(q/11)-coupled muscarinic or purinergic receptors. The effect of SKF81297 could be mimicked by forskolin or 8-Br-cAMP. Further, cholera toxin and the cAMP-dependent protein kinase (PKA) inhibitors, KT5720 and H89, as well as thapsigargin abrogated the D1 receptor evoked Ca(2+) transients. Removal of the priming agonist and treatment with the phospholipase C inhibitor U73122 also blocked the SKF81297-evoked responses. D1R agonist did not stimulate IP(3) production, but pretreatment of cells with the D1R agonist potentiated G(q)-linked receptor agonist mobilization of intracellular Ca(2+) stores. In neurons, SKF81297 and SKF83959, a partial D1 receptor agonist, promoted Ca(2+) oscillations in response to G(q/11)-coupled metabotropic glutamate receptor (mGluR) stimulation. The effects of both D1R agonists on the mGluR-evoked Ca(2+) responses were PKA dependent. Altogether the data suggest that dopamine D1R activation and ensuing cAMP production dynamically regulates the efficiency and timing of IP(3)-mediated intracellular Ca(2+) store mobilization.

Gene linkage and association studies have implicated the region of chromosome 10q containing the calcyon locus with attention deficit hyperactivity disorder (ADHD), bipolar disorder, and schizophrenia susceptibility. In addition, levels of calcyon protein and transcripts are also significantly increased in postmortem tissue from schizophrenic brains. But whether altered calcyon expression might be part of the disease etiology or merely a patho-physiological side effect is not known. To begin to address this issue, we generated a transgenic mouse line (Cal(OE)) using the human calcyon cDNA in which calcyon expression is up-regulated in a number of forebrain structures including the hippocampus, prefrontal cortex (PFC), striatum, and amygdala. Compared to control littermates, the Cal(OE) mice display a range of abnormal behaviors including spontaneous hyperactivity, reduced anxiety, and/or impaired restraint (harm avoidance) that would indicate that calcyon up-regulation leads to deficits in control over behavioral output.

NF-kappaB plays a major role in regulating the immune system. Therefore, alterations in NF-kappaB activity have profound effects on many immunopathologies, including inflammation, autoimmunity, and lymphoid neoplasia. We investigated the effects of estrogen (17beta-estradiol) on NF-kappaB in C57BL/6 mice since estrogen is a natural immunomodulator and we have recently reported that estrogen up-regulates several NF-kappaB-regulated proteins (inducible NO synthase, IFN-gamma, and MCP-1). We found that in vivo estrogen treatment had differential effects on NF-kappaB family members. Estrogen profoundly blocked the nuclear translocation of p65, c-Rel, and Rel-B, partially blocked p52, but permitted translocation of p50. Despite blockade of both the classical (p65/p50) and alternative (RelB/p52) NF-kappaB activation pathways, estrogen induced constitutive NF-kappaB activity and increased the levels of cytokines regulated by NF-kappaB (IL-1 alpha, IL-1 beta, IL-10, and IFN-gamma). Studies involving a NF-kappaB inhibitor confirmed a positive regulatory role of NF-kappaB on these cytokines. Remarkably, estrogen selectively induced B cell lymphoma 3 (Bcl-3), which is known to associate with p50 to confer transactivation capabilities, thereby providing a potential link between observed p50 DNA-binding activity and estrogen up-regulation of NF-kappaB transcriptional activity. Chromatin immunoprecipitation assays confirmed that Bcl-3 bound to the promoter of the NF-kappaB-regulated inducible NO synthase gene in cells from estrogen-treated mice. Estrogen appeared to act at the posttranscriptional level to up-regulate Bcl-3 because mRNA levels in splenocytes from placebo- and estrogen-treated mice were comparable. The novel findings of differential regulation of NF-kappaB proteins by estrogen provide fresh insight into potential mechanisms by which estrogen can regulate NF-kappaB-dependent immunological events.