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Browsing by Author "Heid, Bettina"

Now showing 1 - 11 of 11
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    Deletion of microRNA-183-96-182 Cluster in Lymphocytes Suppresses Anti-DsDNA Autoantibody Production and IgG Deposition in the Kidneys in C57BL/6-Fas(lpr/lpr) Mice
    Wang, Zhuang; Heid, Bettina; Lu, Ran; Sachdeva, Mohit; Edwards, Michael R.; Ren, JingJing; Cecere, Thomas E.; Khan, Deena; Jeboda, Taschua; Kirsch, David G.; Reilly, Christopher M.; Dai, Rujuan; Ahmed, S. Ansar (Frontiers, 2022-07-07)
    Dysregulated miRNAs have been implicated in the pathogenesis of systemic lupus erythematosus (SLE). Our previous study reported a substantial increase in three miRNAs located at the miR-183-96-182 cluster (miR-183C) in several autoimmune lupus-prone mice, including MRL/lpr and C57BL/6-lpr (B6/lpr). This study reports that in vitro inhibition of miR-182 alone or miR-183C by specific antagomirs in activated splenocytes from autoimmune-prone MRL/lpr and control MRL mice significantly reduced lupus-related inflammatory cytokines, interferon-gamma (IFN gamma), and IL-6 production. To further characterize the role of miR-182 and miR-183C cluster in vivo in lupus-like disease and lymphocyte phenotypes, we used hCD2-iCre to generate B6/lpr mice with conditional deletion of miR-182 or miR-183C in CD2(+) lymphocytes (miR-182(-/-)B6/lpr and miR-183C(-/-)B6/lpr). The miR-182(-/-)B6/lpr and miR-183C(-/-)B6/lpr mice had significantly reduced deposition of IgG immunocomplexes in the kidney when compared to their respective littermate controls, although there appeared to be no remarkable changes in renal pathology. Importantly, we observed a significant reduction of serum anti-dsDNA autoantibodies in miR-183C(-/-)B6/lpr mice after reaching 24 weeks-of age compared to age-matched miR-183C(fl/fl)B6/lpr controls. In vitro activated splenocytes from miR-182(-/-)B6/lpr mice and miR-183C(-/-)B6/lpr mice showed reduced ability to produce lupus-associated IFN gamma. Forkhead box O1(Foxo1), a previously validated miR-183C miRNAs target, was increased in the splenic CD4(+) cells of miR-182(-/-)B6/lpr and miR-183C(-/-)B6/lpr mice. Furthermore, in vitro inhibition of Foxo1 with siRNA in splenocytes from miR-182(-/-)B6/lpr and miR-183C(-/-)B6/lpr mice significantly increased IFN gamma expression following anti-CD3/CD28 stimulation, suggesting that miR-182 and miR-183C miRNAs regulate the inflammatory IFN gamma in splenocytes via targeting Foxo1. The deletion of either miR-182 alone or the whole miR-183C cluster, however, had no marked effect on the composition of T and B cell subsets in the spleens of B6/lpr mice. There were similar percentages of CD4(+), CD8(+), CD19(+), as well as Tregs, follicular helper T (T-FH), germinal center B (GCB), and plasma cells in the miR-183C(-/-)B6/lpr and miR-182(-/-)B6/lpr mice and their respective littermate controls, miR-183C(fl/fl)B6/lpr and miR-182(fl/fl)B6/lpr mice. Together, our data demonstrate a role of miR-183C in the regulation of anti-dsDNA autoantibody production in vivo in B6/lpr mice and the induction of IFN gamma in in vitro activated splenocytes from B6/lpr mice.
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    Effects of Experimental Sarcocystis neurona-Induced Infection on Immunity in an Equine Model
    Lewis, S. Rochelle; Ellison, Siobhan; Dascanio, John J.; Lindsay, David S.; Gogal, Robert M.; Werre, Stephen R.; Surendran, Naveen; Breen, Meghan E.; Heid, Bettina; Andrews, Frank M.; Buechner-Maxwell, Virginia A.; Witonsky, Sharon G. (2014)
    Sarcocystis neurona is the most common cause of Equine Protozoal Myeloencephalitis (EPM), affecting 0.5-1% horses in the United States during their lifetimes. The objective of this study was to evaluate the equine immune responses in an experimentally induced Sarcocystis neurona infection model. Neurologic parameters were recorded prior to and throughout the 70-day study by blinded investigators. Recombinant SnSAG1 ELISA for serum and CSF were used to confirm and track disease progression. All experimentally infected horses displayed neurologic signs after infection. Neutrophils, monocytes, and lymphocytes from infected horses displayed significantly delayed apoptosis at some time points. Cell proliferation was significantly increased in S. neurona-infected horses when stimulated nonspecifically with PMA/I but significantly decreased when stimulated with S. neurona compared to controls. Collectively, our results suggest that horses experimentally infected with S. neurona manifest impaired antigen specific response to S. neurona, which could be a function of altered antigen presentation, lack of antigen recognition, or both.
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    EGR2 Deletion Suppresses Anti-DsDNA Autoantibody and IL-17 Production in Autoimmune-Prone B6/lpr Mice: A Differential Immune Regulatory Role of EGR2 in B6/lpr Versus Normal B6 Mice
    Dai, Rujuan; Wang, Zhuang; Heid, Bettina; Eden, Kristin; Reilly, Christopher M.; Ahmed, S. Ansar (Frontiers, 2022-06-15)
    Previous studies have reported that deletion of the transcription factor, early growth response protein 2 (EGR2), in normal C57BL/6 (B6) resulted in the development of lupus-like autoimmune disease. However, increased EGR2 expression has been noted in human and murine lupus, which challenges the notion of the autoimmune suppressive role of EGR2 in B6 mice. In this study, we derived both conditional EGR2-/-B6/lpr and EGR2-/-B6 mice to elucidate the immune and autoimmune regulatory roles of EGR2 in autoinflammation (B6/lpr) versus physiologically normal (B6) conditions. We found that conditional EGR2 deletion increased spleen weight, enhanced T cell activation and IFNγ production, and promoted germinal center B cells and LAG3+ regulatory T cells development in both B6/lpr and B6 mice. Nevertheless, EGR2 deletion also showed strikingly differential effects in these two strains on T lymphocyte subsets profile, Foxp3+ Tregs and plasma cell differentiation, anti-dsDNA autoantibodies and immunoglobulins production, and on the induction of IL-17 in in vitro activated splenocytes. Specifically, EGR2 deletion in B6/lpr mice significantly decreased serum levels of anti-dsDNA autoantibodies, total IgG, IgM, IgG1, and IgG2a with reduced plasma cells differentiation. Furthermore, EGR2 deletion in B6/lpr mice had no obvious effect on IgG immunocomplex deposition, medium caliber vessel, and glomeruli inflammation but increased complement C3 immunocomplex deposition and large caliber vessel inflammation in the kidneys. Importantly, we demonstrated that EGR2 deletion in B6/lpr mice significantly reduced pathogenic CD4-CD8-CD3+B220+ double negative T cells, which correlated with the reduced anti-dsDNA autoantibodies in serum and decreased IL-17 production in splenocytes of EGR2-/-B6/lpr mice. Together, our data strongly suggest that the role of EGR2 is complex. The immunoregulatory role of EGR2 varies at normal or autoinflammation conditions and should not be generalized in differential experimental settings.
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    EGR2 is elevated and positively regulates inflammatory IFNγ production in lupus CD4+ T cells
    Dai, Rujuan; Heid, Bettina; Xu, Xiguang; Xie, Hehuang David; Reilly, Christopher M.; Ahmed, Sattar Ansar (2020-07-09)
    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.
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    Identification of a Common Lupus Disease-Associated microRNA Expression Pattern in Three Different Murine Models of Lupus
    Dai, Rujuan; Zhang, Yan; Khan, Deena; Heid, Bettina; Caudell, David L.; Crasta, Oswald R.; Ahmed, Sattar Ansar (PLOS, 2010-12-10)
    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.
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    Low-dose 17α-ethinyl estradiol (EE) exposure exacerbates lupus renal disease and modulates immune responses to TLR7/9 agonists in genetically autoimmune-prone mice
    Edwards, Michael R.; Dai, Rujuan; Heid, Bettina; Cowan, Catharine; Werre, Stephen R.; Cecere, Thomas E.; Ahmed, Sattar Ansar (Springer Nature, 2020)
    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.
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    Neutrophils and neutrophil serine proteases are increased in the spleens of estrogen-treated C57BL/6 mice and several strains of spontaneous lupus-prone mice
    Dai, Rujuan; Cowan, Catharine; Heid, Bettina; Khan, Deena; Liang, Zhihong; Pham, Christine T.N.; Ahmed, Sattar Ansar (PLOS, 2017-02-13)
    Estrogen, a natural immunomodulator, regulates the development and function of diverse immune cell types. There is now renewed attention on neutrophils and neutrophil serine proteases (NSPs) such as neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (CG) in inflammation and autoimmunity. In this study, we found that although estrogen treatment significantly reduced total splenocytes number, it markedly increased the splenic neutrophil absolute numbers in estrogen-treated C57BL/6 (B6) mice when compared to placebo controls. Concomitantly, the levels of NSPs and myeloperoxidase (MPO) were highly upregulated in the splenocytes from estrogen-treated mice. Despite the critical role of NSPs in the regulation of non-infectious inflammation, by employing NE-/-/PR3-/-/CG-/- triple knock out mice, we demonstrated that the absence of NSPs affected neither estrogen's ability to increase splenic neutrophils nor the induction of inflammatory mediators (IFNγ, IL-1β, IL-6, TNFα, MCP-1, and NO) from ex vivo activated splenocytes. Depletion of neutrophils in vitro in splenocytes with anti-Ly6G antibody also had no obvious effect on NSP expression or LPS-induced IFNγ and MCP-1. These data suggest that estrogen augments NSPs, which appears to be independent of enhancing ex vivo inflammatory responses. Since estrogen has been implicated in regulating several experimental autoimmune diseases, we extended our observations in estrogen-treated B6 mice to spontaneous autoimmune-prone female MRL-lpr, B6-lpr and NZB/WF1 mice. There was a remarkable commonality with regards to the increase of neutrophils and concomitant increase of NSPs and MPO in the splenic cells of different strains of autoimmune-prone mice and estrogen-treated B6 mice. Collectively, since NSPs and neutrophils are involved in diverse pro-inflammatory activities, these data suggest a potential pathologic implication of increased neutrophils and NSPs that merits further investigation.
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    NLRX1 suppresses tumorigenesis and attenuates histiocytic sarcoma through the negative regulation of NF-lambda B signaling
    Coutermarsh-Ott, Sheryl; Simmons, Alysha; Capria, Vittoria; LeRoith, Tanya; Wilson, Justin E.; Heid, Bettina; Philipson, Casandra W.; Qin, Q.; Hontecillas, Raquel; Bassaganya-Riera, Josep; Ting, Jenny P.-Y.; Dervisis, Nikolaos G.; Allen, Irving C. (Impact Journals, 2016-05-31)
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    Noncanonical NF-κB signaling and the essential kinase NIK modulate crucial features associated with eosinophilic esophagitis pathogenesis
    Eden, Kristin; Rothschild, Daniel E.; McDaniel, Dylan K.; Heid, Bettina; Allen, Irving C. (The Company of Biologists, 2017)
    Eosinophilic esophagitis (EoE) is an allergic disease of the esophagus driven by T cell and eosinophil responses to dietary allergens, resulting in chronic mucosal inflammation. Few spontaneous animal models of esophageal eosinophilia exist, with most studies relying on artificial sensitization procedures. NF-κBinducing kinase (NIK; MAP3K14) is a key signaling molecule of the noncanonical NF-κB (NFKB1) pathway, an alternative signaling cascade producing chemokines involved in lymphoid stroma development and leukocyte trafficking. Nik−/− mice have been shown to develop a hypereosinophilic syndrome in peripheral blood and major filtering organs; however, the gastrointestinal mucosa of these mice has not been well characterized. We show that Nik−/− mice develop significant, localized eosinophilic esophagitis that mimics human EoE, including features such as severe eosinophil accumulation, degranulation, mucosal thickening, fibrosis and basal cell hyperplasia. The remainder of the GI tract, including the caudal stomach, small intestine and colon, in mice with active EoE are unaffected, also similar to human patients. Gene expression patterns in esophageal tissue of Nik−/− mice mimics human EoE, with thymic stromal lymphopoetin (TSLP) in particular also elevated at the protein level. In gene expression data sets from human biopsy specimens, we further show that many genes associated with noncanonical NF- κB signaling are significantly dysregulated in EoE patients, most notably a paradoxical upregulation of NIK itself with concurrent upregulation of powerful protein-level destabilizers of NIK. These findings suggest that Nik−/− mice could be useful as a spontaneous model of specific features of EoE and highlight a novel role for noncanonical NF-κB signaling in human patients.
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    Sex differences in the expression of lupus-associated miRNAs in splenocytes from lupus-prone NZB/WF1 mice
    Dai, Rujuan; McReynolds, Savannah; LeRoith, Tanya; Heid, Bettina; Liang, Zhihong; Ahmed, Sattar Ansar (2013-11-01)
    Background 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. Methods 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. Results 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. Conclusion 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.
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    Similar dysregulation of lupus-associated miRNAs in peripheral blood mononuclear cells and splenic lymphocytes in MRL/lpr mice
    Wang, Zhuang; Heid, Bettina; Dai, Rujuan; Ahmed, Sattar Ansar (BMJ, 2018)
    Objective MicroRNAs (miRNAs) play an important role in the pathogenesis of various autoimmune diseases including systemic lupus erythematosus (SLE; lupus). We have previously reported a common pattern of miRNA dysregulation in splenic lymphocytes from several mouse models of lupus. In this study, we investigated whether there is a similar miRNAs expression dysregulation in peripheral blood mononuclear cells (PBMCs) and splenocytes in a classical murine lupus model, MRL/lpr. Method PBMCs were isolated from blood samples of control MRL and lupus MRL/lpr mice aged 14—15 weeks by gradient centrifugation with Histopaque 1083 density media. miRNA TaqMan assays were performed to analyse the expression of 10 lupus-associated miRNAs including miR-182-96-183 cluster, miR-146a, miR-148a, miR-21, miR-31, miR-127, miR-155, and miR-411 in MRL and MRL/lpr PBMCs. Result In this study, we found that 8 out of 10 examined miRNAs (miR-21, miR-31, miR-127, miR-155, miR- 96, miR-182, miR-183 and miR-411) were similarly dysregulated in both PBMCs and splenocytes of MRL/ lpr mice when compared with MRL control mice. Only two miRNAs (miR-146a and miR-148a) showed different dysregulation pattern in the PBMCs and splenocytes of MRL/lpr mice. By comparing with the published miRNA data in human lupus, we demonstrated similarity in miRNA dysregulation in murine and human lupus PBMCs. Conclusion The findings in this study suggest that the miRNA changes observed in PBMCs largely reflect the miRNA dysregulation in cells from the lymphoid organ spleen. Analysis of miRNAs in PBMCs has an advantage over the splenocytes since it allows for monitoring the kinetics of lupus-associated miRNAs expression with peripheral blood cell samples during the development of the disease or after instituting treatment. The similar dysregulation of miRNAs in murine and human lupus PBMCs supports the importance and the feasibility of using murine lupus models to study the pathogenic and therapeutic function of miRNAs in human lupus.