VTechWorks Repository :: Browsing by Author "Reilly, Christopher M."

Browsing by Author "Reilly, Christopher M."

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Altered Germinal-Center Metabolism in B Cells in Autoimmunity
Shiraz, Ashton K.; Panther, Eric J.; Reilly, Christopher M. (MDPI, 2022-01-05)
B lymphocytes play an important role in the pathophysiology of many autoimmune disorders by producing autoantibodies, secreting cytokines, and presenting antigens. B cells undergo extreme physiological changes as they develop and differentiate. Aberrant function in tolerogenic checkpoints and the metabolic state of B cells might be the contributing factors to the dysfunctionality of autoimmune B cells. Understanding B-cell metabolism in autoimmunity is important as it can give rise to new treatments. Recent investigations have revealed that alterations in metabolism occur in the activation of B cells. Several reports have suggested that germinal center (GC) B cells of individuals with systemic lupus erythematosus (SLE) have altered metabolic function. GCs are unique microenvironments in which the delicate and complex process of B-cell affinity maturation occurs through somatic hypermutation (SHM) and class switching recombination (CSR) and where Bcl6 tightly regulates B-cell differentiation into memory B-cells or plasma cells. GC B cells rely heavily on glucose, fatty acids, and oxidative phosphorylation (OXPHOS) for their energy requirements. However, the complicated association between GC B cells and their metabolism is still not clearly understood. Here, we review several studies of B-cell metabolism, highlighting the significant transformations that occur in GC progression, and suggest possible approaches that may be investigated to more precisely target aberrant B-cell metabolism in SLE.
Altered Kinetics of Non-Homologous End Joining Mediated DNA Repair in Mouse Models of Aging and Leukemia
Puthiyaveetil Abdulkader, Abdul Gafoor (Virginia Tech, 2012-09-25)
DNA encodes the genetic instructions for the development and function of organisms and hence maintaining genomic integrity is essential for the propagation of life. However, DNA molecules are under constant threat of metabolic and environmental insults resulting in DNA damages including DNA double strand breaks (DSB), which are considered as a serious threat to cell survival. The majority of these DSB are repaired by Non-homologous end joining (NHEJ). Unrepaired DSB can lead to genomic instability resulting in cell cycle arrest, apoptosis, and mutations. Thus, delineating this DNA repair process is important in understanding the molecular mechanisms of aging and malignant progression. B lymphocytes undergo physiological DNA breaks and NHEJ-mediated DNA repair during their bone marrow differentiation and peripheral class switch recombination (CSR), thus lending them as a good model system in which to delineate the DNA repair mechanisms. To determine the effect of aging on NHEJ, B lymphocytes from old mice were analyzed. The results showed compromised DNA repair in cells from old mice compared to cells from adult mice. These results suggest that NHEJ is compromised during aging and might play critical roles in the aging process and age-associated conditions. To delineate the role of a CT in regulating the immune system, transgenic mice expressing NUP98-HOXD13 (NHD13) were analyzed for B lymphocyte differentiation, peripheral development, CSR, and antibody production. The results showed impaired B cell development and antibody production, which worsened with antigenic stimulation, suggesting the role of NHD13 in immune regulation. These studies explored the possibility of altered NHEJ-mediated DNA repair as a contributing reason for aging process and age-associated conditions. Also, the results from NHD13 study suggested that a primary CT can result in impaired NHEJ and regulate immune cell development and function. Furthermore, the results pointed to the possibility that a primary CT may lead to secondary mutations through altered NHEJ. Thus, these studies shed insight into the molecular mechanisms of altered NHEJ and may help in developing preventive or therapeutic strategies against accumulation of DNA damage, aging process and secondary mutations.
Anti-inflammatory Effects and Biodistribution of Cerium Oxide Nanoparticles
Hirst, Suzanne Marie (Virginia Tech, 2010-02-04)
Cerium oxide nanoparticles have the unique ability to accept and donate electrons, making them powerful antioxidants. Their redox nature is due to oxygen defects in the lattice structure, which are more abundant at the nanoscale. Reactive oxygen species (ROS) are pro-oxidants whose presence is increased during periods of inflammation in the body. ROS damage tissues and cellular function by stripping electrons from proteins, lipids, and DNA. We investigated the ability of nanoceria to quench ROS in vitro and in vivo, and examined the biodistribution and biocompatibility of nanoceria in murine models. Nanoceria was internalized in vitro by macrophages, is non-toxic at the concentrations we investigated, and proteins, mRNA, and oxidative markers of ROS were abated with nanoceria pretreatment in immune stimulated cells as measured by western blot, real time RT PCR, and Greiss assay respectively. In vivo, nanoceria was deposited in the spleen and liver, with trace amounts in the lungs and kidneys as determined by ICP-MS. Using IVIS in vivo imaging, it appeared that nanoceria deposition occurred in lymph tissue. Histology grades show no overt pathology associated with nanoceria deposition, although white blood cell (WBC) counts were generally elevated with nanoceria treatment. Nanoceria suspect particles were seen in lysosomes from kidney samples of IV injected mice in HRTEM images. Lastly, IV nanoceria treatment appears to reduce markers of oxidative stress in mice treated with carbon tetrachloride (CCl4) to induce ROS production. Taken together, our data suggest that nanoceria treatment has the potential to reduce oxidative stress.
Battling Bacteria: Characterizing the NOD-Like Receptor (NLR) Immune Response to Brucella abortus and Borrelia burgdorferi in Host-pathogen Interactions
Tupik, Juselyn D. (Virginia Tech, 2024-08-19)
The innate immune system is integral for defense against infectious diseases. Characterized by Pattern Recognition Receptors (PRRs), which sense conserved molecular motifs known as Pathogen-Associated Molecular Patterns (PAMPs), the innate immune system sets a system of checks and balances to regulate inflammation in host defense. In this dissertation, we focus on one class of PRRs known as the NOD-like Receptors (NLRs) in response to bacterial diseases. This class consists of pro-inflammatory receptors that form a multi-protein complex termed the inflammasome, as well as regulatory NLRs that modulate inflammation. Here, we investigated the roles of inflammasomes and negative regulatory NLRX1 in response to bacterial diseases. First, we studied brucellosis, a zoonotic, chronic disease often transmitted in unpasteurized dairy products from livestock. Using murine models and bone marrow-derived macrophages (BMDMs) challenged with Brucella abortus, we found that canonical inflammasomes in murine models were protective against brucellosis through the initiation of inflammatory cell death called pyroptosis. In contrast, the inhibition of inflammation by NLRX1 adversely led to increased pathology in the spleen and liver in infected murine models. Despite these contrasting results, Brucella genomic DNA was an effective PAMP for NLR recognition. These results suggest the importance of DNA recognition by NLRs during brucellosis. Second, we investigated NLRX1 regulation of Borrelia burgdorferi in Lyme arthritis using murine models. Characterized by persistent inflammation of the joints, Lyme arthritis is an enigmatic and difficult inflammatory condition to resolve. We found that NLRX1 was protective against arthritis. By characterizing changes in gene and protein expression of infected ankle joints, in addition to in vitro studies in BMDMs and fibroblasts, we found that NLRX1 enhances cell migration and regulates cell metabolism. Our results suggest that NLRX1 may metabolically shift macrophages toward a more favorable wound-healing environment for arthritis resolution. Ultimately, this work emphasizes the importance of balance in NLR signaling, which occurs within NLRs and from crosstalk with other inflammatory pathways. Further, NLR signaling is highly multifaceted and context-specific for the cell type and bacterial disease, showcasing the complexity of host-pathogen interactions when battling bacteria.
Catalytic Nanoceria Are Preferentially Retained in the Rat Retina and Are Not Cytotoxic after Intravitreal Injection
Wong, Lily L.; Hirst, Suzanne M.; Pye, Quentin N.; Reilly, Christopher M.; Seal, Sudipta; McGinnis, James F. (PLOS, 2013-03-11)
Cerium oxide nanoparticles (nanoceria) possess catalytic and regenerative radical scavenging activities. The ability of nanoceria to maintain cellular redox balance makes them ideal candidates for treatment of retinal diseases whose development is tightly associated with oxidative damage. We have demonstrated that our stable water-dispersed nanoceria delay photoreceptor cell degeneration in rodent models and prevent pathological retinal neovascularization in vldlr mutant mice. The objectives of the current study were to determine the temporal and spatial distributions of nanoceria after a single intravitreal injection, and to determine if nanoceria had any toxic effects in healthy rat retinas. Using inductively-coupled plasma mass spectrometry (ICP-MS), we discovered that nanoceria were rapidly taken up by the retina and were preferentially retained in this tissue even after 120 days. We also did not observe any acute or long-term negative effects of nanoceria on retinal function or cytoarchitecture even after this long-term exposure. Because nanoceria are effective at low dosages, nontoxic and are retained in the retina for extended periods, we conclude that nanoceria are promising ophthalmic therapeutics for treating retinal diseases known to involve oxidative stress in their pathogeneses.
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.
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.
Developmental Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin: Induced and Exacerbated Autoimmunity in Adulthood
Mustafa, Amjad Issa (Virginia Tech, 2008-12-18)
Developmental 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure can permanently alter immune system ontogeny, resulting in the dysregulation of a number of vital immune pathways. We hypothesized that developmental exposure to TCDD may also impair the establishment of self-tolerance, resulting in an increased risk of autoimmunity. For example, we observed that a single prenatal TCDD exposure given to non-autoimmune-prone high affinity aryl hydrocarbon receptor (AhR) C57BL/6 mice resulted in an immune complex-mediated autoimmune disease during the adult stage. Further using a similar TCDD exposure protocol, autoimmune-prone low affinity AhR SNF1 mice exhibited acceleration and exacerbation of lupus-like nephritis in adulthood. Examination of these mice showed that perinatal TCDD exposure adversely affected both primary immune organs of the adaptive immune system. In the thymic compartment, prenatal TCDD affected thymocyte cellularity, differentiation and maturation as well as central tolerance as indicated by high levels of autoreactive VÎ² TCR T cells in the periphery. Prenatal TCDD also altered bone marrow B lymphopoiesis and B cell maturation and differentiation in the spleen. Functionally, these B cell changes resulted in high serum autoantibodies titers to dsDNA, ssDNA and cardiolipin suggesting a loss in central B cell tolerance. The functional assessment of T cells, via cytokine production showed that prenatal TCDD mice altered Th1/Th2 levels. As a result, significant changes were detected in the kidney characterized by increased immune complex deposition in the glomeruli, lymphocytic infiltration and general pathologic changes. This would suggest that multiple immune pathways are affected by prenatal TCDD and work either independently or synergistically to display immune-mediated disease during aging. Importantly, this study has also shown that the sex of an individual appears to influence both the type of immune pathways affected by TCDD as well as the progression and severity of the autoimmunity. In summary, these studies clearly demonstrate that postnatal immune system impairment due to prenatal TCDD exposure is not limited to immunosuppression but also can include inappropriate immune activation manifested as a hypersensitivity that can lead to the onset of autoimmune disease.
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.
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.
Evaluation of the actin architecture in dysplastic megakaryocytes expressing the NUP98-HOXD13 leukemic fusion gene
Okyere, Benjamin (Virginia Tech, 2013-08-30)
Some myelodysplastic syndrome (MDS) patients present with macrothrombocytopenia due to impaired megakaryocyte (MK) differentiation. Transgenic mice that express the NUP98-HOXD13 (NHD13) fusion gene is a model for MDS and recapitulates the key features of MDS. The study investigated the hypothesis that expression of NHD13 disrupts actin architecture during MK differentiation leading to macrothrombocytopenia. To test the hypothesis, sternums were stained with hematoxylin and eosin, and evaluated by light microscopy to analyze MK morphology in vivo. NHD13 bone marrow (BM) contained many dysplastic MK. BM from wild type (WT) and NHD13 mice were also flushed, cultured in media supplemented with thrombopoietin only or with estrogen to induce proplatelet formation, and MK harvested after 5 days. Harvested MK and BM cores were processed and analyzed by transmission electron microscopy (TEM) to detail the ultrastructural features. TEM of MK revealed that NHD13 leads to formation of an irregular demarcation membrane system and fewer proplatelets. Cultured WT and NHD13 MK were also cytospun onto glass slides, labeled with fluorescent-tagged F-actin, α/β-tubulin and myosin IIa, and their cytoskeleton compared. Interestingly WT MK had actin either distributed evenly or predominantly in the periphery of the cytoplasm, NHD13 MK displayed only the former phenotype. Additionally, proplatelets lacked actin cytoplasmic extensions. The results from the present thesis demonstrate actin expression and architecture are impaired in dysplastic MK expressing the NHD13 leukemic fusion gene and leads to macrothromcytopenia. Understanding the molecular mechanisms of abnormal MK differentiation in MDS is important as many MDS patients die of hemorrhagic complications.
Gut Microbiota and Bacterial DNA Suppress Autoimmunity by Stimulating Regulatory B Cells in a Murine Model of Lupus
Mu, Qinghui; Edwards, Michael R.; Swartwout, Brianna K.; Cabana-Puig, Xavier; Mao, Jiangdi; Zhu, Jing; Grieco, Joseph P.; Cecere, Thomas E.; Prakash, Meeta; Reilly, Christopher M.; Puglisi, Christopher; Bachali, Prathyusha; Grammer, Amrie C.; Lipsky, Peter E.; Luo, Xin M. (2020-11-10)
Autoimmune diseases, such as systemic lupus erythematosus, are characterized by excessive inflammation in response to self-antigens. Loss of appropriate immunoregulatory mechanisms contribute to disease exacerbation. We previously showed the suppressive effect of vancomycin treatment during the "active-disease" stage of lupus. In this study, we sought to understand the effect of the same treatment given before disease onset. To develop a model in which to test the regulatory role of the gut microbiota in modifying autoimmunity, we treated lupus-prone mice with vancomycin in the period before disease development (3-8 weeks of age). We found that administration of vancomycin to female MRL/lpr mice early, only during the pre-disease period but not from 3 to 15 weeks of age, led to disease exacerbation. Early vancomycin administration also reduced splenic regulatory B (Breg) cell numbers, as well as reduced circulating IL-10 and IL-35 in 8-week old mice. Further, we found that during the pre-disease period, administration of activated IL-10 producing Breg cells to mice treated with vancomycin suppressed lupus initiation, and that bacterial DNA from the gut microbiota was an inducer of Breg function. Oral gavage of bacterial DNA to mice treated with vancomycin increased Breg cells in the spleen and mesenteric lymph node at 8 weeks of age and reduced autoimmune disease severity at 15 weeks. This work suggests that a form of oral tolerance induced by bacterial DNA-mediated expansion of Breg cells suppress disease onset in the autoimmune-prone MRL/lpr mouse model. Future studies are warranted to further define the mechanism behind bacterial DNA promoting Breg cells.
Immunomodulation and T Helper TH1/TH2 Response Polarization by CeO2 and TiO2 Nanoparticles
Schanen, Brian C.; Das, Soumen; Reilly, Christopher M.; Warren, William L.; Self, William T.; Seal, Sudipta; Drake, Donald R., III (PLOS, 2013-05-08)
Immunomodulation by nanoparticles, especially as related to the biochemical properties of these unique materials, has scarcely been explored. In an in vitro model of human immunity, we demonstrate two catalytic nanoparticles, TiO2 (oxidant) and CeO2 (antioxidant), have nearly opposite effects on human dendritic cells and T helper (TH) cells. For example, whereas TiO2 nanoparticles potentiated DC maturation that led towards TH1-biased responses, treatment with antioxidant CeO2 nanoparticles induced APCs to secrete the anti-inflammatory cytokine, IL-10, and induce a TH2-dominated T cell profile. In subsequent studies, we demonstrate these results are likely explained by the disparate capacities of the nanoparticles to modulate ROS, since TiO2, but not CeO2 NPs, induced inflammatory responses through an ROS/inflammasome/IL-1β pathway. This novel capacity of metallic NPs to regulate innate and adaptive immunity in profoundly different directions via their ability to modulate dendritic cell function has strong implications for human health since unintentional exposure to these materials is common in modern societies.
The Impact of Protein Acetylation/Deacetylation on Systemic Lupus Erythematosus
Ren, Jingjing; Panther, Eric J.; Liao, Xiaofeng; Grammer, Amrie C.; Lipsky, Peter E.; Reilly, Christopher M. (MDPI, 2018-12-12)
Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease in which the body’s immune system mistakenly attacks healthy cells. Although the exact cause of SLE has not been identified, it is clear that both genetics and environmental factors trigger the disease. Identical twins have a 24% chance of getting lupus disease if the other one is affected. Internal factors such as female gender and sex hormones, the major histocompatibility complex (MHC) locus and other genetic polymorphisms have been shown to affect SLE, as well as external, environmental influences such as sunlight exposure, smoking, vitamin D deficiency, and certain infections. Several studies have reported and proposed multiple associations between the alteration of the epigenome and the pathogenesis of autoimmune disease. Epigenetic factors contributing to SLE include microRNAs, DNA methylation status, and the acetylation/deacetylation of histone proteins. Additionally, the acetylation of non-histone proteins can also influence cellular function. A better understanding of non-genomic factors that regulate SLE will provide insight into the mechanisms that initiate and facilitate disease and also contribute to the development of novel therapeutics that can specifically target pathogenic molecular pathways.
Inhibition of Heat Shock Protein 90 Reduces Inflammatory Signal Transduction in Murine J774 Macrophage Cells and Lessens Disease in Autoimmune MRL/lpr Mice: What in vitro, in vivo, and in silico Models Reveal
Shimp, Samuel Kline (Virginia Tech, 2012-04-30)
Heat shock protein 90 (HSP90) is a molecular chaperone protein that protects proteins from degradation, repairs damaged proteins, and assists proteins in carrying out their functions. HSP90 has hundreds of clients, many of which are inflammatory signaling kinases. The mechanism by which HSP90 enables inflammatory pathways is an active area of investigation. The HSP90 inhibitors such as geldanamycin (GA) and its derivative 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) have been shown to reduce inflammation. It was hypothesized that inhibiting HSP90 would reduce inflammatory signal cascade levels. To test this, J774 mouse macrophage cells were treated with 17-DMAG and immune-stimulated with lipopolysaccharide (LPS). 17-DMAG treatment reduced nitric oxide (NO) production and the expression of pro-inflammatory cytokines interleukin (IL)-6, IL-12, and TNF-α. Inhibition of HSP90 also prevented nuclear translocation of NF-κB. To investigate the anti-inflammatory effects of HSP90 inhibition in vivo, MRL/lpr lupus mice were administered 5 mg/kg 17-DMAG for six weeks via intraperitoneal injection. Mice treated with 17-DMAG were found to have reduced proteinuria and reduced splenomegaly. Flow cytometric analysis of splenocytes showed that 17-DMAG decreased double negative T (DNT) cells. Renal expression of HSP90 was also measured and found to be increased in MRL/lpr mice that did not receive 17-DMAG. The mechanistic interactions between HSP90 and the pro-inflammatory nuclear factor-κB (NF-κB) pathway were studied and a computational model was developed. The model predicts cellular response of inhibitor of κB kinase (IKK) activation and NF-κB activation to LPS stimulation. Model parameters were fit to IKK activation data. Parameter sensitivity was assessed through simulation studies and showed a strong dependence on IKK-HSP90 binding. The model also accounts for the effect of a general HSP90 inhibitor to disrupt the IKK-HSP90 interaction for reduced activation of NF-κB. Model simulations were validated with experimental data. In conclusion, HSP90 facilitates inflammation through multiple signal pathways including Akt and IKK. Inhibition of HSP90 by 17-DMAG reduced disease in the MRL/lpr lupus mouse model. A computational model supported the hypothesis that HSP90 is required for IKK to activate the NF-κB pathway. Taken together, HSP90 is a prime target for therapeutic regulation of many inflammatory processes and warrants further study to understand its mechanism of regulating cell signaling cascades.
Isoform-Selective HDAC Inhibition for the Treatment of Lupus Nephritis
Regna, Nicole Lynn (Virginia Tech, 2014-06-19)
Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease requiring a genetic predisposition coupled with an environmental trigger in order for initiation of disease. While the exact pathoaetiology has yet to be determined, both B and T cell dysregulation are thought to contribute to disease. Histone deacetylases (HDACs) are a class of enzymes that hydrolyze the lysine bound acetyl group in both histone and non-histone proteins thereby altering protein structure and function. While the use of pan-HDAC inhibitors has proven to be effective for the treatment of a number of acute diseases, they may not be viable as therapeutics for chronic disease due to cytotoxicity and adverse side effects following long term treatment. We sought to determine whether treatment with a class I and II HDAC inhibitor (HDACi) or a specific HDAC6i would be able to ameliorate disease in lupus-prone NZB/W mice. We found that both the class I and II HDACi (ITF2357) and the HDAC6i (ACY-738) were able to decrease SLE markers of disease including splenomegaly, proteinuria, and anti-dsDNA and IgG production in the sera. Treatment with ITF2357 resulted in an increase in the number of immunosuppressive regulatory T (Treg) cells and a decrease in the pro-inflammatory Th17 phenotype. Furthermore, ITF2357 was found to increase Foxp3 acetylation leading to increased Foxp3 stability allowing for differentiation into the Treg phenotype. ACY-738 treatment was able to correct aberrant bone marrow B cell differentiation while also increasing the number of splenic Treg cells in NZB/W mice. These results suggest that HDAC inhibition is able to ameliorate SLE in NZB/W mice by altering aberrant T and B cell differentiation. Additional studies were performed to further examine the expression and function of different HDAC isoforms in immune cells. Due to the ability of HDAC inhibition to decrease markers of SLE disease as well as alter B and T cell development and differentiation, we sought to determine if specific HDAC isoforms are altered in lupus vs non lupus mice in early and late disease states. We determined the level of class IIb HDAC (HDACs 6, 9, and 10) expression in bone marrow B cells, splenic B and T cells, and glomerular cells from early- and late-disease MRL/lpr lupus-prone mice compared to healthy, age-matched C57BL/6 control mice. Expression of HDAC6 and HDAC9 were significantly increased in all of the tissues tested from MRL/lpr mice. Furthermore, both cytoplasmic and nuclear HDAC activity was increased in diseased MRL/lpr mice, and HDAC activity and expression continued to increase as disease progressed. In vitro treatment with ACY-738, a selective HDAC6i, was able to decrease cytoplasmic HDAC activity and inhibit iNOS production. Furthermore, ACY-738 was able to alter apoptosis through increased Bax expression in B cells. Treatment with ACY-738 was also able to inhibit Hsp90 expression and decrease NF-κB nuclear translocation, which are both upregulated during active SLE. Our studies indicate that HDAC activity contributes to SLE pathogenesis and that the use of isoform-selective HDAC inhibitors may be a viable treatment for SLE.
Lactobacillus spp. act in synergy to attenuate splenomegaly and lymphadenopathy in lupus-prone MRL/lpr mice
Cabana-Puig, Xavier; Mu, Qinghui; Lu, Ran; Swartwout, Brianna; Abdelhamid, Leila; Zhu, Jing; Prakash, Meeta; Cecere, Thomas E.; Wang, Zhuang; Callaway, Sabrina; Sun, Sha; Reilly, Christopher M.; Ahmed, S. Ansar; Luo, Xin M. (Frontiers, 2022-07-28)
Commensal bacteria and the immune system have a close and strong relationship that maintains a balance to control inflammation. Alterations of the microbiota, known as dysbiosis, can direct reactivity to self-antigens not only in the intestinal mucosa but also at the systemic level. Our laboratory previously reported gut dysbiosis, particularly lower abundance of bacteria in the family Lactobacillaceae, in lupus-prone MRL/lpr mice, a model of systemic autoimmunity. Restoring the microbiota with a mix of 5 different Lactobacillus species (spp.), L. reuteri, L. oris, L. johnsonii, L. gasseri and L. rhamnosus, attenuated lupus-liked clinical signs, including splenomegaly and lymphadenopathy. However, our understanding of the mechanism was limited. In this study, we first investigated the effects of individual species. Surprisingly, none of the species individually recapitulated the benefits of the mix. Instead, Lactobacillus spp. acted synergistically to attenuate splenomegaly and renal lymphadenopathy through secreted factors and a CX3CR1-dependent mechanism. Interestingly, oral administration of MRS broth exerted the same benefits likely through increasing the relative abundance of endogenous Lactobacillus spp. Mechanistically, we found increased percentages of FOXP3-negative type 1 regulatory T cells with administration of the mix in both spleen and mesenteric lymph nodes. In addition, oral gavage of Lactobacillus spp. decreased the percentage of central memory T cells while increasing that of effector memory T cells in the lymphoid organs. Furthermore, a decreased percentage of double negative T cells was observed in the spleen with the mix. These results suggest that Lactobacillus spp. might act on T cells to attenuate splenomegaly and lymphadenopathy. Together, this study advances our understanding of how Lactobacillus spp. attenuate lupus in MRL/lpr mice. The synergistic action of these bacteria suggests that multiple probiotic bacteria in combination may dampen systemic autoimmunity and benefit lupus patients.
Leaky Gut As a Danger Signal for Autoimmune Diseases
Mu, Q.; Kirby, J.; Reilly, Christopher M.; Luo, Xin M. (Frontiers, 2017-05-23)
The intestinal epithelial lining, together with factors secreted from it, forms a barrier that separates the host from the environment. In pathologic conditions, the permeability of the epithelial lining may be compromised allowing the passage of toxins, antigens, and bacteria in the lumen to enter the blood stream creating a “leaky gut.” In individuals with a genetic predisposition, a leaky gut may allow environmental factors to enter the body and trigger the initiation and development of autoimmune disease. Growing evidence shows that the gut microbiota is important in supporting the epithelial barrier and therefore plays a key role in the regulation of environmental factors that enter the body. Several recent reports have shown that probiotics can reverse the leaky gut by enhancing the production of tight junction proteins; however, additional and longer term studies are still required. Conversely, pathogenic bacteria that can facilitate a leaky gut and induce autoimmune symptoms can be ameliorated with the use of antibiotic treatment. Therefore, it is hypothesized that modulating the gut microbiota can serve as a potential method for regulating intestinal permeability and may help to alter the course of autoimmune diseases in susceptible individuals.
MicroRNA-mediated Attenuation of Inflammation in NZB/W Lupus Mice
Chafin, Cristen Brooke (Virginia Tech, 2013-10-08)
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production and deposition of nuclear self-antigen-containing immune complexes. Epigenetic factors, including altered microRNA (miRNA) expression, may contribute to aberrant immune cell function in SLE. miRNAs are small, noncoding RNAs that bind to the 3’ untranslated region of target mRNAs resulting in post-transcriptional gene modulation. IL-6, an inflammatory cytokine overproduced by mesangial cells in SLE, contains a potential binding site for miR-let-7a. In order to examine if alterations in miR-let-7a expression can influence inflammatory mediator production in SLE, we isolated mesangial cell miRNAs from 8 and 32- week-old female New Zealand Black/White (NZB/W) mice. We found miR-let-7a expression was significantly increased in the mesangial cells of pre-diseased and actively diseased NZB/W mice compared to age-matched female New Zealand White (NZW) controls. Overexpression of miR-let-7a in vitro increased IL-6 production in LPS/IFN-γ-stimulated mesangial cells compared to the stimulated control. Due to the crucial role of miR-let-7a in cell division and inflammation, we investigated miR-let-7a-mediated proliferation and NFκB activation in J774A.1 macrophages and MES 13 mesangial cells in vitro. Cell proliferation, retinoblastoma protein (Rb) phosphorylation, and NFκB activation were increased in cells transfected with miR-let-7a and stimulated with LPS/IFN-γ. Expression and production of the cell cycle inhibitor E2F5 was decreased in stimulated cells overexpressing miR-let-7a. We found that the cell cycle promoter E2F2 and NFκB target the miR-let-7a promoter. Next we sought to determine alterations in iii specific disease-associated miRNAs in female NZB/W mice treated with hydroxychloroquine (HCQ) or prednisone (PRED) for 12 weeks beginning at 20 weeks-of-age. We found that treatment with HCQ or PRED induced unique changes to miRNA expression in multiple tissues. In order to identify specific miRNAs as disease-modifying agents and not merely disease correlates, further in vitro analyses confirmed HCQ or PRED-mediated inhibition of miRNAs is critical to alter the inflammatory response. Taken together, our results suggest that overexpression of miR-let-7a may contribute to hyperplasia and the proinflammatory response in SLE. Our studies indicate that lupus therapeutics may work, in part, by altering the expression of disease-associated miRNAs in immune cells and the urine.
MicroRNAs Implicated in the Immunopathogenesis of Lupus Nephritis
Chafin, Cristen B.; Reilly, Christopher M. (Hindawi, 2013-07-07)
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the deposition of immune complexes due to widespread loss of immune tolerance to nuclear self-antigens. Deposition in the renal glomeruli results in the development of lupus nephritis (LN), the leading cause of morbidity and mortality in SLE. In addition to the well-recognized genetic susceptibility to SLE, disease pathogenesis is influenced by epigenetic regulators such as microRNAs (miRNAs). miRNAs are small, noncoding RNAs that bind to the 3′ untranslated region of target mRNAs resulting in posttranscriptional gene modulation. miRNAs play an important and dynamic role in the activation of innate immune cells and are critical in regulating the adaptive immune response. Immune stimulation and the resulting cytokine milieu alter miRNA expression while miRNAs themselves modify cellular responses to stimulation. Here we examine dysregulated miRNAs implicated in LN pathogenesis from human SLE patients and murine lupus models. The effects of LN-associated miRNAs in the kidney, peripheral blood mononuclear cells, macrophages, mesangial cells, dendritic cells, and splenocytes are discussed. As the role of miRNAs in immunopathogenesis becomes delineated, it is likely that specific miRNAs may serve as targets for therapeutic intervention in the treatment of LN and other pathologies.

Browsing by Author "Reilly, Christopher M."

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