VTechWorks Repository :: Browsing by Author "Ahmed, Sattar Ansar"

Browsing by Author "Ahmed, Sattar Ansar"

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Antibiotics ameliorate lupus-like symptoms in mice
Mu, Qinghui; Tavella, Vincent J.; Kirby, Jay L.; Cecere, Thomas E.; Chung, Matthias; Lee, Jiyoung; Li, Song; Ahmed, Sattar Ansar; Eden, Kristin; Allen, Irving C. (Nature, 2017-10-20)
Gut microbiota and the immune system interact to maintain tissue homeostasis, but whether this interaction is involved in the pathogenesis of systemic lupus erythematosus (SLE) is unclear. Here we report that oral antibiotics given during active disease removed harmful bacteria from the gut microbiota and attenuated SLE-like disease in lupus-prone mice. Using MRL/lpr mice, we showed that antibiotics given after disease onset ameliorated systemic autoimmunity and kidney histopathology. They decreased IL-17-producing cells and increased the level of circulating IL-10. In addition, antibiotics removed Lachnospiraceae and increased the relative abundance of Lactobacillus spp., two groups of bacteria previously shown to be associated with deteriorated or improved symptoms in MRL/lpr mice, respectively. Moreover, we showed that the attenuated disease phenotype could be recapitulated with a single antibiotic vancomycin, which reshaped the gut microbiota and changed microbial functional pathways in a time-dependent manner. Furthermore, vancomycin treatment increased the barrier function of the intestinal epithelium, thus preventing the translocation of lipopolysaccharide, a cell wall component of Gram-negative Proteobacteria and known inducer of lupus in mice, into the circulation. These results suggest that mixed antibiotics or a single antibiotic vancomycin ameliorate SLE-like disease in MRL/lpr mice by changing the composition of gut microbiota.
Characterization of basal and lipopolysaccharide-induced microRNA expression in equine peripheral blood mononuclear cells using Next-Generation Sequencing
Parkinson, Nicholas J.; Buechner-Maxwell, Virginia A.; Witonsky, Sharon G.; Pleasant, R. Scott; Werre, Stephen R.; Ahmed, Sattar Ansar (PLOS, 2017-05-26)
The innate immune response to lipopolysaccharide contributes substantially to the morbidity and mortality of gram-negative sepsis. Horses and humans share an exquisite sensitivity to lipopolysaccharide and thus the horse may provide valuable comparative insights into this aspect of the inflammatory response. MicroRNAs, small non-coding RNA molecules acting as post-transcriptional regulators of gene expression, have key roles in toll-like receptor signaling regulation but have not been studied in this context in horses. The central hypothesis of this study was that lipopolysaccharide induces differential microRNA expression in equine peripheral blood mononuclear cells in a manner comparable to humans. Illumina Next Generation Sequencing was used to characterize the basal microRNA transcriptome in isolated peripheral blood mononuclear cells from healthy adult horses, and to evaluate LPS-induced changes in microRNA expression in cells cultured for up to four hours. Selected expression changes were validated using quantitative reverse-transcriptase PCR. Only miR-155 was significantly upregulated by LPS, changing in parallel with supernatant tumor necrosis factor-α concentration. Eight additional microRNAs, including miR-146a and miR-146b, showed significant expression change with time in culture without a clear LPS effect. Target predictions indicated a number of potential immunity-associated targets for miR-155 in the horse, including SOCS1, TAB2 and elements of the PI3K signaling pathway, suggesting that it is likely to influence the acute inflammatory response to LPS. Gene alignment showed extensive conservation of the miR-155 precursor gene and associated promoter regions between horses and humans. The basal and LPS-stimulated microRNA expression pattern characterized here were similar to those described in human leukocytes. As well as providing a resource for further research into the roles of microRNAs in immune responses in horses, this will facilitate inter-species comparative study of the role of microRNAs in the inflammatory cascade during endotoxemia and sepsis.
Control of lupus nephritis by changes of gut microbiota
Mu, Qinghui; Zhang, Husen; Liao, Xiaofeng; Lin, Kaisen; Liu, Hualan; Edwards, Michael R.; Ahmed, Sattar Ansar; Yuan, Ruoxi; Li, Liwu; Cecere, Thomas E.; Branson, David B.; Kirby, Jay L.; Goswami, Poorna; Leeth, Caroline M.; Read, Kaitlin A.; Oestreich, Kenneth J.; Vieson, Miranda D.; Reilly, Christopher M.; Luo, Xin M. (2017-07-11)
Background: Systemic lupus erythematosus, characterized by persistent inflammation, is a complex autoimmune disorder with no known cure. Immunosuppressants used in treatment put patients at a higher risk of infections. New knowledge of disease modulators, such as symbiotic bacteria, can enable fine-tuning of parts of the immune system, rather than suppressing it altogether. Results: Dysbiosis of gut microbiota promotes autoimmune disorders that damage extraintestinal organs. Here we report a role of gut microbiota in the pathogenesis of renal dysfunction in lupus. Using a classical model of lupus nephritis, MRL/lpr, we found a marked depletion of Lactobacillales in the gut microbiota. Increasing Lactobacillales in the gut improved renal function of these mice and prolonged their survival. We used a mixture of 5 Lactobacillus strains (Lactobacillus oris, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus johnsonii, and Lactobacillus gasseri), but L. reuteri and an uncultured Lactobacillus sp. accounted for most of the observed effects. Further studies revealed that MRL/lpr mice possessed a “leaky” gut, which was reversed by increased Lactobacillus colonization. Lactobacillus treatment contributed to an anti-inflammatory environment by decreasing IL-6 and increasing IL-10 production in the gut. In the circulation, Lactobacillus treatment increased IL-10 and decreased IgG2a that is considered to be a major immune deposit in the kidney of MRL/lpr mice. Inside the kidney, Lactobacillus treatment also skewed the Treg-Th17 balance towards a Treg phenotype. These beneficial effects were present in female and castrated male mice, but not in intact males, suggesting that the gut microbiota controls lupus nephritis in a sex hormone-dependent manner. Conclusions: This work demonstrates essential mechanisms on how changes of the gut microbiota regulate lupusassociated immune responses in mice. Future studies are warranted to determine if these results can be replicated in human subjects.
Effects of estrogen on the B cell functions of normal mice
Verthelyi, Daniela I. (Virginia Tech, 1996)
It is now recognized that reproductive hormones such as estrogen influence not only classical targets (eg. reproductive tissues), but may also act on non-classical target sites such as the immune system. A better understanding of the effects of estrogen on the immune system is of paramount importance since: (i) increasing numbers of women around the world take estrogen-containing oral contraceptives, some times for most of their reproductive life; (ii) estrogen is often prescribed as a replacement therapy to postmenopausal women; and (iii) a large number of pesticides, insecticides, and phytoestrogens (plant-derived estrogens) have been found to have hormone disrupting effects as evidenced by altered development of reproductive and immune functions in wild species. The precise effects of estrogen on the normal immune system are not well known. The overall objective of this work has been to better understand the role of sex hormones on the B cell function of normal mice. It is hoped that this will lead to an improved understanding of the pathogenesis and treatment of immune-related disorders such as autoimmune diseases and cancer. These studies were accomplished by parenteral administration of estrogen to nonautoimmune C57BL/6 mice, a widely used strain in immunology. In these mice, we found that treatment with estrogen, but not 5a-dihydrotestosterone. induced the expression of a wide variety of IgG and IgM autoantibodies and heteroantibodies that are associated with autoimmune and infectious diseases. These include antibodies to cardiolipin and other membrane phospholipids, dsDNA and acetone-killed Brucella abortus strain RB51. Importantly, the expression of anti-dsDNA and anti-cardiolipin antibodies was sustained for several months after the removal of the exogenous source of estrogen. This indicates that the immunomodulatory effect of estrogen is long-lasting. These antibodies have a marginal degree of crossreactivity with other antigens and belong mainly to IgG2b subisotype. These findings were confirmed at the cellular level, where we have shown that estrogen-treated mice have increased numbers of plasma cells in the spleen, and that these plasma cells actively secrete IgM and IgG immunoglobulins as assessed by ELISPOT. Further, higher immunoglobulin yield per cell was evident in estrogen-treated than in placebo-treated controls in the spleen and bone marrow. Interestingly, we found that splenic lymphocytes had an increase in antibody-forming cells for all specificities tested. Active antibody-forming cells from bone marrow preferentially recognized autoantigens, cardiolipin and dsDNA. Functional analysis on the viability of the splenic lymphocytes showed that in vivo exposure to estrogen resulted in: (a) increase in the proportion of cells dying by apoptosis, and (b) an increased proportion of lymphocytes that were actively proliferating as assessed by cell cycle analysis. Culturing of B cells in the absence of any deliberate stimulus showed the B cells from estrogen-treated mice underwent active proliferation and resisted death by apoptosis more compared to controls. We also found that despite the autoproliferative character of splenic B cells, they were able to respond adequately to stimulation with anti-CD40 antibodies, IL-4 and lipopolysaccharides. B cells from mice treated with estrogen had a marked reduction in their susceptibility to apoptosis when cultured in the presence of such stimuli. Together these studies indicate that normal mice exposed to estrogen may express a variety of autoantibodies, show signs of B cell hyperactivity, have defects in susceptibility of B cells to apoptosis as well as the ability to proliferate in the absence of stimulation. It is hoped that these studies would enhance our understanding of the immunomodulatory role of estrogen in health and in a wide range of disorders such as autoimmune and cancer disorders.
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.
Epigenetic Contribution and Genomic Imprinting Dlk1-Dio3 miRNAs in Systemic Lupus Erythematosus
Dai, Rujuan; Wang, Zhuang; Ahmed, Sattar Ansar (MDPI, 2021-05-01)
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.
Estrogen up-regulates inducible nitric oxide synthase, nitric oxide, and cyclooxygenase-2 in splenocytes activated with T cell stimulants: Role of interferon-gamma
Karpuzoglu, Ebru; Fenaux, Jillian B.; Phillips, Rebecca A.; Lengi, Andrea J.; Elvinger, Francois; Ahmed, Sattar Ansar (Endocrine Society, 2006-02)
Estrogen is implicated in many autoimmune diseases and is a robust immunomodulator. For example, it regulates interferon (IFN)-gamma, a cytokine believed to up-regulate inducible nitric oxide synthase (iNOS). A notable gap in the literature is a lack of information on the regulation of nitric oxide in immune tissues by estrogen. We now show that activation of splenocytes with T cell stimulants [concanavalin-A (Con-A) or anti-CD3 antibodies] results in copious release of nitric oxide in splenocyte cultures from estrogen-treated but not placebo-treated mice. Moreover, even a low dose of T cell stimulants induced nitric oxide in splenocytes from estrogen-treated, but not placebo-treated, mice. Con-A-activated splenocytes from estrogen-treated mice also have up-regulated iNOS mRNA, iNOS protein, and cyclooxygenase-2 (a nitric oxide-regulated downstream proinflammatory protein) when compared with controls. Our studies suggest that the induction of nitric oxide by activated splenocytes from estrogen-treated mice is mediated in part by IFN gamma. First, blocking costimulatory signals mediated through interactions of CD28 and B7 molecules by CTLA-4Ig markedly decreased not only IFN gamma but also nitric oxide. Second, estrogen treatment of IFN gamma-knockout (IFN gamma(-)/(-)) mice did not induce iNOS protein or nitric oxide. Finally, in vitro addition of recombinant IFN gamma to Con-A-activated splenocytes from IFN gamma((-)/(-)) mice induced iNOS protein primarily in estrogen-treated mice. Overall, this is the first report to show that estrogen treatment up-regulates IFN gamma-inducible-iNOS gene expression, iNOS protein, nitric oxide, and cyclooxygenase-2 as an indirect consequence of activation of T cells. These findings may have wide implications to immunity and inflammatory disorders including female-predominant autoimmune diseases.
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.
Intracellular growth of Brucella abortus and B. melitensis in murine macrophage-like cell lines and partial characterization of a biologically active extract from B. abortus strain RB51
Wise, Darla J. (Virginia Tech, 1995)
Brucella abortus is a gram negative, facultative intracellular bacterial pathogen, capable of growth and replication within macrophages, and is the causative agent of bovine brucellosis. The progression of brucellosis within the host is determined by the interaction of Brucella with its host. Therefore, it was of interest to specifically examine several features of the Brucella-host interaction. The Brucella-macrophage interaction is central in the progression of brucellosis and, therefore, it was possible to study this interaction in vitro in the form of the abilities of Brucella strains to grow and replicate within macrophages. Furthermore, it was of interest to see if the in vitro model was capable of assessing the degree of attenuation of the Brucella. Various strains of B. abortus and B. melitensis were used to infect two murine macrophage-like cell lines to study their intracellular growth kinetics and to compare these kinetics with the growth characteristics observed in mice. It was determined that Brucella growth in one murine macrophage-like cell line (J774.A1) clearance pattern reflected the in vivo growth kinetics of the various Brucella tested. All strains tested in the macrophage model had a significant 1-4 log decrease in intracellular bacteria at 24 hours post infection. The decrease in intracellular numbers at 24 hours postinfection was due to the bactericidal activities of the macrophages as opposed to changes in the Brucella. This model was determined not a satisfactory means in vitro for assessing the degree of attenuation of Brucella mutants, as the model was not capable of predicting the reduced virulence of the B. melitensis RM1 rifampin resistant mutant. Vaccination with live B. abortus strain RB51 protects both mice and cattle against challenge with virulent strain 2308. As B. abortus is a facultative intracellular pathogen, the host's cell-mediated immunity is assumed to be important in the clearance of the bacteria. Therefore, selected antigens from strain RB51 could be used and tested in vitro for their ability to induce a cellular immune, specifically a T helper type 1 (T_H1) response, by splenocytes from mice vaccinated with strain RB51. An extract from strain RB51, designated S2, was found to stimulate the proliferation of splenic lymphocytes from strain RB51 vaccinated mice as well as the production of interleukin (IL)-2 and interferon (INF)-γ, but not IL-4. This cytokine profile is consistent with a cell-mediated T_H1 immune response. As the T_H1 response is assumed to be important in the clearance of Brucella by the host, mice were immunized with S2 extract in adjuvant. Upon virulent challenge with strain 2308, no protection was observed as compared with challenged control mice. The S2 extract contained nine proteins ranging in size from 10 kDa to 80 kDa. In order to determine which of the individual S2 proteins was responsible for the observed T_H1 activity, a means of separating the proteins into individual bands for testing was needed. Sufficient resolution of the nine proteins could not be accomplished by isoelectric focusing (Rotofor) or by fast performance liquid chromatography (FPLC). Therefore, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate S2 into individual proteins. Two proteins, 10.2 kDa and 11.5 kDa in size, were observed to stimulate the production of INF-γ by sensitized splenocytes. The N-terminal amino acid sequences of these two proteins were obtained and their putative DNA sequences deduced. Another antigen known to be a component of the S2 extract is the lipopolysaccharide (LPS). Polymyxin B is thought not to bind B. abortus LPS as it does not contain 3-myristic acid. However, it was important to evaluate the role, if any, of LPS contamination of the S2 extract in the in vitro responses observed with splenocytes from mice vaccinated with strain RB51. The S2 extract was treated with polymyxin B linked to agarose beads. The treated S2 extract contained less 2-keto-3- deoxyoctonate (KDO) which indirectly suggests a decrease in LPS. Furthermore, the proliferation of sensitized splenocytes induced by polymyxin B treated S2 extract was observed to decrease slightly, while the INF-γ levels were observed to increase following the treatment. Reactivity of a monoclonal antibody (Bru 48) specific for B. abortus rough LPS was observed by immunoblot analysis only with samples not treated with polymyxin B beads. This suggests that the amount of rough B. abortus LPS contained in the preparation is less than is detectable by the monoclonal antibody when the S2 extract was treated with polymyxin B beads. In contrast to published results, these data suggest that polymyxin B interacts with B. abortus LPS. The use of a live vector delivery system as a means of vaccination needs to be examined further in the case of brucellosis. In this manner, the expression of the S2 proteins may more accurately be assessed as to their role in eliciting a protective immune response.
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.
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.
Our Environment Shapes Us: The Importance of Environment and Sex Differences in Regulation of Autoantibody Production
Edwards, Michael R.; Dai, Rujuan; Ahmed, Sattar Ansar (Frontiers, 2018-03-08)
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.
Paradoxical Effects of All-Trans-Retinoic Acid on Lupus-Like Disease in the MRL/lpr Mouse Model
Liao, Xiaofeng; Ren, Jingjing; Wei, Cheng-Hsin; Ross, A. Catharine; Cecere, Thomas E.; Jortner, Bernard S.; Ahmed, Sattar Ansar; Luo, Xin M. (PLOS, 2015-03-16)
Roles of all-trans-retinoic acid (tRA), a metabolite of vitamin A (VA), in both tolerogenic and immunogenic responses are documented. However, how tRA affects the development of systemic autoimmunity is poorly understood. Here we demonstrate that tRA have paradoxical effects on the development of autoimmune lupus in the MRL/lpr mouse model. We administered, orally, tRA or VA mixed with 10% of tRA (referred to as VARA) to female mice starting from 6 weeks of age. At this age, the mice do not exhibit overt clinical signs of lupus. However, the immunogenic environment preceding disease onset has been established as evidenced by an increase of total IgM/IgG in the plasma and expansion of lymphocytes and dendritic cells in secondary lymphoid organs. After 8 weeks of tRA, but not VARA treatment, significantly higher pathological scores in the skin, brain and lung were observed. These were accompanied by a marked increase in B-cell responses that included autoantibody production and enhanced expression of plasma cell-promoting cytokines. Paradoxically, the number of lymphocytes in the mesenteric lymph node decreased with tRA that led to significantly reduced lymphadenopathy. In addition, tRA differentially affected renal pathology, increasing leukocyte infiltration of renal tubulointerstitium while restoring the size of glomeruli in the kidney cortex. In contrast, minimal induction of inflammation with tRA in the absence of an immunogenic environment in the control mice was observed. Altogether, our results suggest that under a predisposed immunogenic environment in autoimmune lupus, tRA may decrease inflammation in some organs while generating more severe disease in others.
Regulation of IL-17 in autoimmune diseases by transcriptional factors and microRNAs
Khan, Deena; Ahmed, Sattar Ansar (Frontiers, 2015-07-14)
In recent years, IL-17A (IL-17), a pro-inflammatory cytokine, has received intense attention of researchers and clinicians alike with documented effects in inflammation and autoimmune diseases. IL-17 mobilizes, recruits and activates different cells to increase inflammation. Although protective in infections, overproduction of IL-17 promotes inflammation in autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, psoriasis, among others. Regulating IL-17 levels or action by using IL-17-blocking antibodies or IL-17R antagonist has shown to attenuate experimental autoimmune diseases. It is now known that in addition to IL-17-specific transcription factor, ROR gamma t, several other transcription factors and select microRNAs (miRNA) regulate IL-17. Given that miRNAs are dysregulated in autoimmune diseases, a better understanding of transcriptional factors and miRNA regulation of IL-17 expression and function will be essential for devising potential new therapies. In this review, we will overview IL-17 induction and function in relation to autoimmune diseases. In addition, current findings on transcriptional regulation of IL-17 induction and plausible interplay between IL-17 and miRNA in autoimmune diseases are highlighted.
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.
Signal Transducer and Activation of Transcription (STAT) 4 beta, a Shorter Isoform of Interleukin-12-Induced STAT4, Is Preferentially Activated by Estrogen
Karpuzoglu, Ebru; Phillips, Rebecca A.; Dai, Rujuan; Graniello, Carmine; Gogal, Robert M.; Ahmed, Sattar Ansar (Endocrine Society, 2009-03)
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)
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.
Tumor-induced macrophage and T cell dysfunction
Walker, Thomas M. (Virginia Tech, 1995)
Macrophages (MΦ) and T cells mediate helper, effector, and cytotoxic activities. Tumor growth changes the phenotypic and functional characteristics of MΦ and T cells and shifts them toward suppressor phenotypes and functional activities. Tumor growth changed MΦ DNA synthesis when activated through Mac-1 and Mac-3 surface molecules, which suggests that specific receptor-ligand interactions modulate MΦ cell-cycle kinetics differently in the tumor-bearing host (TBH). Tumor growth changed MΦ responsiveness to MΦ colony-stimulating factor (M-CSF). M-CSF did not reverse decreases in autorecognition caused by TBH MΦ, and increased TBH MΦ suppression during T-cell alloreactivity. TBH suppressor activities were associated predominantly with MHC class II MΦ. TBH class II⁻ MΦ quantitatively and qualitatively suppressed T-cell autoreactivity partly by dysregulation of interferon-gamma (IFN-y), interleukin-4 (IL-4), and prostaglandin E₂ (PGE₂) production. TBH MΦ had aberrant regulation of granulocyte-macrophage colony-stimulating factor (GM-CSF). TBH MΦ produced less GM-CSF than normal host (NH) MΦ. GM-CSF failed to increase class II molecule expression on TBH MΦ, and TBH class II⁻ MΦ became more suppressive when cultured with GM-CSF. TBH MΦ GM-CSF dysregulation involved PGE₂ and interleukin-10 (IL-10). Tumor growth also affected CD4⁺ and CD8⁺ T cell phenotype and function. The in vivo percentage of CD8⁺ T cells significantly increased during tumor growth and these cells significantly suppressed T-cell allorecognition and autorecognition. TBH CD8⁺ T-cell suppression was mediated partly through dysregulation of IFN-y, IL-4, and PGE₂ production. TBH CD4⁺ T cells produced less GM-CSF than NH CD4⁺ T cells, and GM-CSF dysregulation was linked partly to increased sensitivity to IL-10 and transforming growth factor-β₁ (TGF-β₁). Tumor growth changed CD4⁺ T cell responsiveness to cytokines associated with T cell activation. TBH CD4⁺ T cell proliferation was suppressed significantly by taxol. This research also suggests that taxol can promote tumor regression. Taxol disrupted tumor cell growth through cytostatic and cytotoxic mechanisms, and increased tumor cell susceptibility to taxol-induced, MΦ-derived lytic molecules. Taxol also disrupted autocrine regulation of TGF-β₁ and stimulated apoptosis. Collectively, these studies suggest that tumor-associated changes in MΦ and T cells involve cytokine dysregulation. |Immunotherapeutic approaches may partly or completely reverse suppressor immune cell activities during tumor growth.
The Upregulation of Genomic Imprinted DLK1-Dio3 miRNAs in Murine Lupus Is Associated with Global DNA Hypomethylation
Dai, Rujuan; Lu, Ran; Ahmed, Sattar Ansar (PLOS, 2016-04-12)
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.

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