Elattar, Sawsan; Department of Biochemistry and Molecular Biology / Cancer Center (8/7/2018)
      SAWSAN ELATTAR The Tumor Secretory Factor ZAG Promotes White Adipose Tissue Browning and Energy Wasting in Cachexia (Under the direction of SATYANARAYANA ANDE) Cachexia is a complex tissue-wasting syndrome characterized by inflammation, hyper-metabolism, increased energy expenditure and anorexia. Browning of white adipose tissue (WAT) is one of the significant factors that contribute to energy wasting in cachexia. Tumors secrete an array of secretory factors, such as tumor necrosis factor α (TNFα), interleukin-1 (IL-1), interleukin-6 (IL-6), interferon γ (IFNγ) and zinc-α2-glycoprotein (ZAG), that have been implicated in altering metabolism and promoting cachexia. Previous studies have demonstrated that ZAG can induce lipolysis; however, whether ZAG plays a role beyond lipolysis remains unclear. Here, by utilizing a cell implantation model, we demonstrate that the lipid-mobilizing factor, ZAG, induces WAT browning in mice. Increased circulating levels of ZAG not only induced lipolysis in the adipose tissues, but also caused robust browning in the WAT. Stimulating white adipose progenitors with ZAG recombinant protein or expression of ZAG in mouse embryonic fibroblasts (MEFs) strongly enhanced brown-like differentiation. At the molecular level, ZAG stimulated peroxisome proliferator-activated receptor gamma (PPARƔ) and early B cell factor 2 (Ebf2) expression and promoted their recruitment to the PR/SET domain 16 (Prdm16) promoter, leading to enhanced expression of Prdm16, which determines brown cell fate. In the brown adipose tissue (BAT), ZAG stimulated the expression of PPARƔand peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) and promoted recruitment of PPARƔ to the uncoupling protein 1 (UCP1) promoter, leading to increased expression of UCP1. Collectively, by promoting WAT browning and by activating thermogenesis in the BAT, ZAG increased body energy expenditure. Overall, our results revealed a novel function of ZAG in WAT browning and highlight that targeting ZAG may have therapeutic applications in humans with cachexia. KEYWORDS: (Cachexia, beige adipocyte, brown fat, adipose atrophy, Zinc-α2-glycoprotein, Ebf2, Prdm16, PPARƔ, UCP1)
    • Canonical Wnt Signaling in Antigen Presenting Cells Regulates Microbiota-Induced Inflammation and Immune Cell Homeostasis in the Colon

      Swafford, Daniel Joseph; Department of Biochemistry and Molecular Biology / Cancer Center (8/3/2018)
      Aberrant Wnt/β-catenin-signaling occurs in several inflammatory diseases including inflammatory bowel disease (IBD) and IBD-associated colon carcinogenesis. However, its role in shaping mucosal immune responses to commensals in the gut remains unknown. Here, we investigated the importance of canonical Wnt signaling in CD11c+ antigen presenting cells (APCs) in controlling intestinal inflammation. Using a mouse model of ulcerative colitis, we demonstrated that canonical Wnt-signaling in intestinal CD11c+ antigen presenting cells (APCs) controls intestinal inflammation by imparting an anti-inflammatory phenotype. Genetic deletion of Wnt co-receptors, low-density lipoprotein receptor-related protein 5 and 6 (LRP5/6) in CD11c+ APCs in mice (LRP5/6ΔCD11c mice) resulted in enhanced intestinal inflammation with increased histopathological severity of colonic tissue. This was due to microbiota-dependent increased production of pro-inflammatory cytokines and decreased expression of immune regulatory factors such as IL-10, retinoic acid (RA), and IDO. In addition, loss of LRP5/6-mediated signaling in CD11c+ APCs resulted in altered microflora and T cell homeostasis, which led to a loss of systemic tolerance to oral antigen. Furthermore, our study demonstrates that conditional activation of β-catenin in CD11c+ APCs in LRP5/6ΔCD11c mice resulted in reduced acute intestinal inflammation with decreased histopathological severity of colonic tissue. Loss of canonical Wnt signaling in CD11c+ APCs also results in an increase in colonic polyp formation and exacerbation of chronic inflammation/injury. This was also heavily dependent on the presence and composition of the gut microbiota, as fecal transfers from LRP5/6ΔCD11c mice to floxed control (LRP5/6FL/FL) mice that were administered an antibiotic cocktail produces a polyp load and weight loss similar to that of LRP5/6ΔCD11c mice without treatment. Additionally, our study demonstrates that conditional activation of β-catenin in CD11c+ APCs in LRP5/6ΔCD11c mice reduces severity of inflammation-associated colon carcinogenesis in these mice. Furthermore, we show that treatment of LRP5/6ΔCD11c mice with either RA or IL-10 reduces severity of inflammation-associated colon carcinogenesis. Mechanistically, RA and IL-10 may independently reduce key inflammatory factors at the acute phase of colitis. These results ultimately reveal a mechanism by which intestinal APCs control intestinal inflammation and immune homeostasis via the canonical Wnt signaling pathway, which may serve as a promising target for chronic inflammatory disorders.
    • Polyfunctional CD4+ T cells synergize with chemotherapy to reprogram tumor metabolism towards a curative outcome

      Habtetsion, Tsadik Ghebreamlak; Department of Biochemistry and Molecular Biology (8/3/2017)
      CD4+ T cells are critical mediators of anti-tumor immunity. Accumulating evidence from preclinical and clinical studies suggests that tumor-reactive CD4+ T cells in adoptive T cell therapy (ACT) have the potential to effectively control tumor growth. In most ACT clinical settings, chemotherapeutic agents are used to induce an immunostimulatory milieu which facilitates the effector function of donor T cells. Although the efficacy of ACT has been well-established, currently only a fraction of patients with certain types of malignancy have benefited, highlighting the need for improved ACT strategies. Recent studies have revealed that the metabolic reprogramming by cancer cells attenuate antitumor immune response by imposing nutrient restrictions in the tumor microenvironment, which leads to defective T cell responses. In the current study, we set out to explore how Cyclophosphamide (CTX) and tumor reactive CD4+ T cells alter the metabolic features of cancer cells. By comparing the global metabolic profiling of tumors pre and post-treatment, we found that CXT+CD4 ACT elicited a metabolic catastrophe affecting multiple pathways critical for cancer progression. Particularly, CTX+CD4 ACT led to marked reduction in glutathione (GSH) levels, increased accumulation of reactive oxygen species (ROS) and oxidative DNA damage product in tumors. Importantly, administration of N-acetyl-L-cysteine diminished the curative effect of CTX+CD4 ACT. Moreover, pharmacological inhibition of GSH using Buthionine Sulfoximine (BSO) following CTX significantly delayed tumor growth in mice. Mechanistically, we found that TNFα synergized with chemotherapy to reduce intracellular GSH levels and promote ROS induction. TNFα enhanced cell death in chemotherapy pre-treated tumor cells and the cytotoxic effect was reversed by adding GSH exogenously. Importantly, the curative effect of CTX+CD4 ACT was abrogated after TNFα neutralization. Additionally, we found that CTX+CD4 ACT led to tumor vascular disruption causing hemorrhagic necrosis of tumors. IFNγR-deficient mice failed to reject tumor after CTX+CD4 ACT and had intact tumor vasculature. Collectively, our data reveal that tumor reactive CD4+T cells disrupt the redox homeostasis of cancer cells in TNFα-dependent manner. Whereas CD4+T cells derived IFNγ targeted tumor endothelial cells to cause vascular disruption and tissue ischemia. The combined action of these two cytokines leads to eventual eradication of established tumor.

      Angara, Kartik Prasad; Department of Biochemistry and Molecular Biology (Augusta University, 7/20/2018)
      Glioblastoma (GBM) is a hypervascular and hypoxic neoplasia of the central nervous system with an extremely high rate of mortality. Owing to its hypervascularity, anti-angiogenic therapies (AAT) have been used as an adjuvant to the traditional surgical resection, chemotherapy, and radiation to normalize blood vessels, control abnormal vasculatures and prevent recurrence. The benefits of AAT have been transient and the tumors were shown to relapse faster and demonstrated particularly high rates of AAT-induced therapy resistance due to activation of alternative neovascularization mechanisms. Vascular Mimicry (VM) is the uncanny ability of tumor cells to acquire endothelial-like properties, lay down vascular patterned networks reminiscent of host endothelial blood vessels and served as an irrigation system for the tumors to meet with the increasing metabolic and nutrient demands in the event of the ensuing hypoxia resulting from AAT. In our studies, we have demonstrated that AAT accelerates VM. We observed that Vatalanib (a VEGFR2 tyrosine kinase inhibitor) induced VM vessels are positive for periodic acid-Schiff (PAS) matrix but devoid of any endothelium on the inner side and lined by tumor cells on the outer side. Interestingly, 20-HETE synthesis inhibitor HET0016 significantly decreased GBM tumors through decreasing VM structures both at the core and at the periphery of the tumors. During our extensive studies to understand the tumor-inherent mechanisms of AAT-induced resistance, we identified a crucial chemokine, CXCL8 or IL-8, to be highly upregulated in the GBM tumors treated with AAT. IL-8 has been well established as a highly prevalent cytokine in GBM with potent pro-migratory and pro-angiogenic functions. AAT-treated groups had significantly higher populations of CXCR2+ glioma stem cells and endothelial-like subpopulations and these populations were decreased following treatment with HET0016 and SB225002 (a CXCR2 antagonist). CXCR2+ GBM tumor cells were shown to form VM-like vascular channels carrying functional RBCs. Knocking down CXCR2 led to smaller tumor size in the animals and improperly developed vascular structures without CXCR2+ GBM cells lining them. This confirms our hypothesis that CXCR2+ GBM cells initiate VM and contribute to AAT resistance in GBM. Our present study suggests that HET0016 and SB225002 have potential to target therapeutic resistance and can be combined with other antitumor agents in preclinical and clinical trials.
    • Super enhancer-associated molecular signatures reveal a dependency on immune and metabolic mechanisms in chronic lymphatic leukemia

      Shull, Austin Y.; Department of Biochemistry and Molecular Biology (7/12/2016)
      Chronic lymphocytic leukemia (CLL), characterized by the progressive and uncontrolled accumulation of CD19+ B cells, currently remains as an incurable malignancy despite recent advancements in treatment options. The difficulties of eliciting curative measures in CLL are partly driven by the adaptability of the transcriptional response in CLL cells. In this study, we sought to better understand the complexities of the CLL transcriptional profile by defining the large histone H3 lysine-27 acetylation regions known as “super enhancers” within B cells and determining which genes overexpressed in CLL overlapped with super enhancers. From this analysis, we identified 190 super enhancer-associated genes overexpressed in CLL and determined that many of the genes identified were either involved in immune signaling cascades (e.g. LCK, FCER2) or metabolic regulation (e.g. LSR, ENO2). These processes corresponded with our reverse phase protein array (RPPA) profile of CLL patients, which shows overexpression of immune signaling kinases (e.g. LCK) as well as alteration of metabolically sensitive translation regulators (e.g. 4E-BP1 phosphorylation). Additionally, we determined that CLL cells are apoptotically sensitive to dual PI3K/mTOR inhibition when compared to upstream B cell receptor pathway inhibition due to their differential effects on 4E-BP1 phosphorylation. Based on the derived information from our super enhancer expression signature, we then compared the effects of preferentially targeting super enhancers with either the BET bromodomain inhibitor JQ1 or the cyclin dependent kinase-7 (CDK7) inhibitor THZ1. From this comparison, we saw that JQ1 could inhibit cell cycle progression in CLL cell lines as well as differentially disrupt transcription of genes involved in immune signaling. Contrastingly, we saw that THZ1 elicited a different response in CLL cells by inducing apoptosis and differentially downregulating genes involved in metabolism. The specific super enhancer-associated genes disrupted by the respective treatments further highlighted the dichotomy of JQ1 and THZ1-mediated effects, as JQ1 suppressed the B cell activation marker gene FCER2 whereas THZ1 suppressed the glycolytic enolase gene ENO2. Collectively, these results reveal that super enhancers play a role in mediating both immune signaling and metabolic expression signatures in CLL and that super enhancers can be differentially disrupted by BET bromodomain or CDK7 inhibition.

      Elmasry, Khaled; Department of Biochemistry and Molecular Biology / Cancer Center (5/22/2018)
      Our earlier studies have established the role of 12/15-lipoxygenase (LO) in mediating the inflammatory reaction in diabetic retinopathy. However, the exact mechanism is still unclear. The goal of the current study was to identify the potential role of endoplasmic reticulum (ER) stress as a major cellular stress response in the 12/15-LO-induced retinal changes in diabetic retinopathy. We used in vivo and in vitro approaches. For in vivo studies, experimental diabetes was induced in wild-type (WT) mice and 12/15-Lo (also known as Alox15) knockout mice (12/15-Lo−/−); ER stress was then evaluated after 12-14 weeks of diabetes. We also tested the effect of intravitreal injection of 12-hydroxyeicosatetraenoic acid (HETE) on retinal ER stress in WT mice and in mice lacking the catalytic subunit of NADPH oxidase, encoded by Nox2 (also known as Cybb) (Nox2−/− mice). In vitro studies were performed using human retinal endothelial cells (HRECs) treated with 15-HETE (0.1 µmol/l) or vehicle, with or without ER stress or NADPH oxidase inhibitors. This was followed by evaluation of ER stress response, NADPH oxidase expression/activity and the levels of phosphorylated vascular endothelial growth factor receptor-2 (p-VEGFR2) by western blotting and immunoprecipitation assays. Moreover, real-time imaging of intracellular calcium (Ca2+) release in HRECs treated with or without 15-HETE was performed using confocal microscopy. Deletion of 12/15-Lo significantly attenuated diabetes-induced ER stress in mouse retina. In vitro, 15-HETE upregulated ER stress markers such as phosphorylated RNA-dependent protein kinase-like ER-regulated kinase (p-PERK), activating transcription factor 6 (ATF6) and protein disulfide isomerase (PDI) in HRECs. Inhibition of ER stress reduced 15-HETE-induced-leukocyte adhesion, VEGFR2 phosphorylation and NADPH oxidase expression/activity. However, inhibition of NADPH oxidase or deletion of Nox2 had no effect on ER stress induced by the 12/15-LO-derived metabolites both in vitro and in vivo. We also found that 15-HETE increases the intracellular calcium in HRECs. ER stress contributes to 12/15-LO-induced retinal inflammation in diabetic retinopathy via activation of NADPH oxidase and VEGFR2. Perturbation of calcium homeostasis in the retina might also play a role in linking 12/15-LO to retinal ER stress and subsequent microvascular dysfunction in diabetic retinopathy.
    • The Differential Roles of PI3K P110 Isoforms in Regulating CD4 T Cell Subset Polarization

      Webb, Mason James; Department of Biochemistry and Molecular Biology (5/12/2017)
      Class IA phosphatidylinositol-4,5-bisphosphate 3-kinases, or PI3K’s, are one of the earliest bottlenecks for T cell receptor signaling transduction, without which phosphorylated phosphatidylinositides cannot be generated and the T cell activation cascade becomes impaired. Of the catalytic class IA PI3K subunits, there are three isoforms designated as p110α, p110β, and p110δ. The Khleif laboratory has discovered that these catalytic subunits display unique roles in T regulatory cells and non-polarized activated CD4+ T cells. This thesis aims to determine what differential control these p110 isoforms have upon distinct polarized CD4+ T cell subsets.

      Abdelrahman, Ammar; Department of Biochemistry and Molecular Biology (Augusta University, 2020-12)
      Currently, treatments of diabetic retinopathy (DR) have limited therapeutic benefits and limited accessibility to the growing diabetic population at risk because of the high expenses and complicated procedures. Inflammation, endothelial dysfunction, and microvascular damage are common features of diabetic complications including DR. GPR109A is the metabolite sensing receptor of beta-hydroxybutyrate (BHB) the principal ketone body in humans. Our previous studies have shown the role of GPR109A expression in promoting anti-inflammatory response in retinal pigmented epithelial (RPE) cells and the relevance of the receptor in DR. Expression of the GPR109A in microvascular endothelial cells (ECs) has been reported recently. However, the relevance of GPR109A expression and activation to retinal EC functions are yet to be studied. Our goal in this study was to identify the role of GPR109A expression and activation in barrier and angiogenic functions of retinal ECs in context of diabetic retinopathy. We used electrical cell impedance sensing (ECIS) technology to evaluate barrier functions in primary human retinal endothelial cells (HRECs) which constitute the inner BRB. Knocking down GPR109A in HRECs with siRNA decreased the transendothelial electrical resistance (TEER) compared to scrambled siRNA. Treating HRECs with BHB increased their TEER and counteracted VEGF-induced barrier disruption through activation of GPR109A and increasing zonula occludens-1 (ZO-1) expression. Treatment of STZ-diabetic mice with exogenous BHB for one month protected against the pathologic albumin leakage induced by diabetes and improved the visual acuity of this animal model of diabetes. Using the mouse model of oxygen induced retinopathy (OIR), we showed that Gpr109a-/- mice had slower vascular recovery from pathologic angiogenesis compared to age matched wild type mice. Moreover, physiologic revascularization of vaso-oblitrated retinas was impaired by loss of GPR109a and associated with dysregulated inflammatory and angiogenic signaling. Collectively, these data point to a role for GPR109A in the regulation of barrier and angiogenic mechanisms in retinal ECs and, promote the receptor as a potential druggable target for impacting these mechanisms in microvascular retinal diseases such as DR.
    • A Molecular Basis of Chemoresistance in Bladder Cancer

      Lahorewala, Sarrah; Biochemistry and Cancer Biology (Augusta University, 2020-12)
      Background: In advanced bladder cancer (BC), development of resistance to the frontline chemotherapeutic drugs Gemcitabine and Cisplatin contributes to the poor prognosis of patients. Newly discovered chondroitinase, HYAL-4 V1 (V1), drives malignant transformation in BC. We evaluated V1’s role and the downstream molecules involved in the mechanistic regulation of chemoresistance in BC. Experimental Design: HYAL-4 expression was evaluated by RT-qPCR and IHC in metastatic muscle-invasive BC patients who received Gemcitabine plus Cisplatin chemotherapy. HYAL-4 wild-type and V1 were stably expressed or silenced in three BC and one normal urothelial cell line. Transfectants were analyzed for Gemcitabine and Cisplatin sensitivity, and for Gemcitabine influx and efflux to determine the mechanism of Gemcitabine resistance. The effect of cytidine deaminase (CDA) inhibition on Gemcitabine sensitivity was evaluated in vitro and in xenograft models. Results: HYAL-4 expression was an independent predictor of disease-specific mortality and treatment failure in our clinical cohort, and stratified patients into higher risk for both those outcomes. V1-expressing BC and normal urothelial cells were resistant to Gemcitabine due to the upregulation of cytidine deaminase (CDA) expression and activity, resulting in increased Gemcitabine metabolism and efflux; treating cells with tetrahydrouridine (THU), a CDA inhibitor, abrogated the chemotherapeutic resistance. Gemcitabine-resistant V1 cells demonstrated increased expression of V1’s substrate CD44 and phosphorylated STAT3. Si-RNA mediated CD44 knockdown and STAT3 inhibition both sensitized cells to Gemcitabine in vitro. In xenograft models, treatment with a combination of Gemcitabine and THU completely inhibited tumor growth. Conclusions: This project discovered V1 as a novel determinant of Gemcitabine resistance and potential predictor of treatment response in BC. V1 drives resistance to Gemcitabine through CD44-STAT3 mediated upregulation of CDA, and inhibiting this pathway sensitizes tumor cells to the therapy in preclinical models of BC.

      Klement, John; Department of Biochemistry and Molecular Biology (Augusta University, 2020-05)
      The host adaptive immune system functions to discriminate self from non-self, eliminating threats from viral infection to tumors. Cytotoxic lymphocytes (CTLs) are the primary effector arm of adaptive immunity. To prevent aberrant activation and autoimmunity, immune checkpoints function physiologically to restrain the CTL response. Tumors pathologically express these checkpoints, preventing immune-driven tumor clearance. Accordingly, immune checkpoint inhibitors (ICIs) have shown remarkable clinical success. However, many types of malignancies, as well as many individual patients with responsive tumor types, fail to benefit from current ICI immunotherapies. This conundrum suggests that as-yet undiscovered immune checkpoints exist. We observed that mice deficient in the transcription factor interferon regulatory factor eight (IRF8) tolerated allogenic tumor grafts and demonstrated impaired CTL activation with an accumulation of CD44hi memory-like CTLs. We sought to investigate the mechanism of this immunosuppression. Conditional deletion of IRF8 in T cells, as well as a mixed chimera model, demonstrated that IRF8 did not directly control CTL activation or differentiation into a CD44hi population. Instead, global loss of IRF8 lead to an expansion of an immature myeloid CD11b+Ly6G+Ly6Clo population which highly expressed osteopontin (OPN), a physiological ligand for CD44. Elevated levels of OPN were shown to suppress murine CTL activation and proliferation. A similar IRF8-OPN-CD44 axis was observed in murine and human colorectal cancer, which is refractory to current ICI therapies. Malignant cells and human patients displayed enhanced OPN levels relative to healthy donor controls. This was shown to be mediated by loss of IRF8 expression, which directly bound to the OPN promoter to repress its transcription. Elevated levels of OPN similarly prevented human CTL activation, and higher levels of OPN were correlated with decreased survival in human patients. We have shown that the IRF8-OPN-CD44 axis functions as a novel immune checkpoint in both myeloid and tumor cells. Blockade of OPN may have potent anti-tumor activity, expanding the pool of patients responsive to ICI therapy.

      Morera, Daley S; Department of Biochemistry and Molecular Biology (Augusta University, 2020-05)
      There is a need for novel prognostic biomarkers and targeted treatments in bladder cancer (BC), even more so for muscle-invasive disease (MIBC). Discovery of molecular markers to predict outcome in BC patients may lead to identification of impactful therapeutic targets. The hyaluronic acid (HA) family of molecules and distinct molecular subtypes have both been investigated as potential prognostic markers. HA family and chondroitin sulfate proteoglycans, such as CD44, have been implicated in driving aggressiveness of disease; however, a chondroitinase enzyme that cleaves chondroitin sulfate proteoglycans has not been identified in any eukaryotic system. We evaluated molecular markers of BC and the first known eukaryotic/human chondroitinase, that we identified, for their ability to predict clinical outcome in patients, and for their roles as drivers of disease. We also investigated the anti-tumor effects of HA synthesis inhibitor 4-methylumbelliferone (4MU), a non-toxic orally bioavailable supplement. This study demonstrates that transcript levels of HA family members can predict metastasis and poor survival in BC patients. HA- family expression also correlated with epithelial mesenchymal transition (EMT) markers β -Catenin, Twist, Snail, and E-Cadherin. HA signaled through its receptors CD44/RHAMM and the PI3-K/AKT axis. 4MU targeted HA signaling, inhibiting proliferation and motility/invasion, inducing apoptosis in vitro, and preventing tumor growth in vivo. We discovered that a previously unidentified splice variant of HYAL-4 was elevated in bladder tumors. We named this variant "V1". Our studies showed that V1 had chondroitinase activity, cleaved chondroitin-6-sulfate from CD44, and consequently increased CD44 secretion. In vivo, V1-expressing urothelial cells formed muscle-invasive tumors and V1-expressing cancer cells developed metastatic tumors. Evaluation of the prognostic significance of the molecular subtypes of MIBC that were recently identified by other groups, showed the subtypes to have little to no predictive ability for clinical outcome in multivariate analyses that included standard clinical parameters. Consistently, clinical parameters such as histopathologic tumor grade, T-stage, and lymph node status, outperformed the molecular subtypes. Contrarily, V1 levels could independently predict metastasis and survival with high efficacy, suggesting that focusing on V1 as a functional biomarker may be a better strategy to improve clinical outcome of BC patients.
    • Gene regulation by the putative Campylobacter jejuni diguanylate cyclase CbrR

      Fulmer, Claudia; Department of Biochemistry and Cancer Biology (Augusta University, 2020-05)
      As a leading cause of bacterial gastroenteritis, Campylobacter jejuni incurs health care costs estimated at $290 million a year in the US and up to 40,000 deaths in children aged 5 and younger worldwide. As such, determining those proteins that regulate C. jejuni virulence factors are prime targets to possibly develop a prophylactic therapy, that as of yet does not exist. CbrR is a C. jejuni response regulator that is annotated as a diguanylate cyclase (DGC), the class of enzyme that catalyzes the synthesis of cyclic di-GMP, a universal bacterial second message molecule, from GTP. In C. jejuni strain DRH212, an unmarked deletion mutant, cbrR-, and complemented mutant, cbrR+, were constructed. Soft agar motility tests, biofilm formation assays, transmission electron microscopy (TEM), and scanning electron microscopy (SEM) were performed. Site-directed mutagenesis was performed on cbrR to make alanine substitutions in both the autoinhibitory site (I-site) and active site and differential radial capillary of action ligand (DRaCALA) assays were performed to determine nucleotide binding by wild-type CbrR and the CbrR point mutants. Soft agar motility assays indicated a hyper-motile phenotype associated with the C. jejuni cbrR- mutant, whereas motility was all but negated in the cbrR+ complement. Biofilm assays and SEM demonstrated similar formation and robustness of biofilms between wild type and cbrR- mutant, however cbrR+ was unable to form significant biofilms in 72 hours. TEM images showed similar cellular morphology between cbrR-, wild type, and cbrR+, however cbrR+ cells had fewer flagella. DRaCALA assays showed wild-type CbrR and the active site mutant were both able to bind GTP and cyclic di-GMP, whereas the I-site mutant lost the ability to bind cyclic di-GMP, indicating the product binding site on CbrR. The highly conserved diguanylate cyclase CbrR is the only annotated DGC in the C. jejuni genome. Though the active site sequence is highly variant when compared to the consensus sequence, this protein is able to bind both substrate and product of the chemical synthesis of cyclic di-GMP and has now been shown to be a negative regulator of motility, a critical virulence factor in C. jejuni pathogenesis.
    • Role of Ufl1 in CD8+ Memory T Cell Survival and Function

      Bhatt, Brinda Nikhil; Department of Biochemistry and Cancer Biology (Augusta University, 2020-05)
      Immunity mediated by CD8+ T cells plays an integral part of the response to eradicate both infections and tumor cells. The recognition of antigen by the T cell receptor triggers a cascade of signaling events in naïve CD8+ T cells that leads to their proliferation and differentiation into memory and effector cells. Cytotoxic effector CD8+ T cells produce large amount of cytokines and effector molecules that play a critical role in the elimination of pathogens. Following pathogen clearance, a small population of memory T cells persists long-term, which has the ability to expand robustly upon re-exposure to antigen and provide the host with rapid and specific recall responses against the pathogen. Treatments boosting an individual’s own immune response have changed the landscape of cancer research. However there are still limitations with patient responsiveness, toxic side effects, and not achieving long-term remission. As advances in immunotherapy revolutionize cancer treatment, understanding the molecular networks governing CD8+ T cell function has become more important than ever. In our studies, we found that genetic knockout of UFM1 Specific Ligase 1 (Ufl1) in T cells leads to a CD8-specific loss of central memory cells. Flow cytometry staining revealed that the population of CD8+CD122+ T cells are significantly reduced in mice lacking Ufl1 in T cells. We observed that Ufl1 deficiency leads to apoptosis of these cells in vivo. Ufl1-deficient CD8+CD122+ T cells express higher amounts of Fas on the cell surface as well as activated cleaved caspase 3. RNA-sequencing analysis demonstrated that these cells also overexpress numerous genes associated with exhaustion, including PD-1, Lag3, Tim3, and 2B4. Interestingly, the Listeria monocytogenes disease model showed that Ufl1-deficient CD8+ T cells behave similarly to wild type cells during the acute effector response, but undergo a more dramatic contraction and subsequently launch an attenuated recall response. Consistent with this, a melanoma-specific vaccine failed to protect mice lacking Ufl1 in T cells. Our present study suggests that Ufl1 plays a critical role in the suppression of apoptosis and exhaustion of memory CD8+ T cells.
    • Primary Tumor-Induced Immunity Is Suppressed By Surgery-Induced Inflammation In The Presence Of Residual Tumor Cells

      Piranlioglu, Raziye; Department of Biochemistry and Molecular Biology (Augusta University, 2019-12)
      It is widely thought that tumor cells disseminate from a primary site into the circulation during the early stages of tumor development. However, the fate of these early disseminated tumor cells (DTCs) has been elusive. By utilizing the murine mammary tumors, 4T1 and EMT6, in a syngeneic mouse model, we show that both tumors disseminate into secondary organs but only 4T1 tumors are able to generate metastasis. In contrast, EMT6 primary tumors induce an anti-tumor response that leads to elimination of DTCs. This anti-tumor immunity is CD8+ T cell-dependent and provides long-term immunity. Furthermore, the mice are free of DTCs within a couple of days when primary tumors are completely resected and they reject subsequently injected tumors, whereas mice with residual tumors following surgery show enhanced local recurrence and outgrowth of DTCs at metastatic sites; this effect may be explained by elevated levels of granulocyte colony-stimulating factor (G-CSF). This increase is accompanied by an accumulation of immature myeloid-derived suppressor cells (MDSCs) in the spleen and lungs, the main target organ for metastasis. Moreover, the infiltration of a granulocytic subset of MDSCs (gMDSCs) leads to a decrease in a subset of T cells that have a role in long-term immunity. Our goal for this study is to elucidate how immune components of distant organs affect the fate of DTCs and the role of surgery induced-inflammation in generating a pre-metastatic niche. Our studies may also provide a molecular explanation of improved overall survival in breast cancer patients following complete resection of primary tumors with negative margins.

      Naughton, Sean X; Biomedical Sciences (Augusta University, 2019-12)
      Organophosphates (OPs) are a broad class of chemicals with a variety of uses that include pesticides, chemical warfare agents, fuel additives, and plasticizers. Due to their sheer number of applications and known toxicological profile, OPs represent a persistent concern to human health worldwide. Furthermore, the effects of OPs that occur independently of their well-known mechanism of acute toxicity (AChE inhibition) have not been well studied. The presented research seeks to expand upon our understanding of AChE-independent mechanisms of OP toxicity as well as to identify potential therapies for treating these negative effects. In Manuscript 1 we demonstrate that the OP diisopropylfluorophosphate (DFP) induced axonal transport deficits occur in vivo at exposure levels that were not associated with cholinergic toxicity. Additionally, we observed deficits in white matter integrity following sub-acute DFP exposure. In Manuscript 2 we present a series of experiments, which were conducted to identify potential therapeutic compounds for the treatment of OP induced deficits in axonal transport. Here, we utilized a phenotypic drug-screening assay in order to identify compounds that could be protective against DFP. In Manuscript 3 we present data which demonstrates that the carbamate physostigmine does not impair axonal transport, as has been previously demonstrated with OPs. These experiments were critical to demonstrating the AChE independence of OP-induced axonal transport deficits and further elucidate the unique nature of OP toxicity in comparison to other AChE inhibitors. Collectively, these studies contribute to a better understanding of the full spectrum of toxicological effects of OPs and provide insightful findings into potential therapeutics for the treatment of OP related toxicity.

      Ajith, Ashwin; Biomedical Sciences (Augusta University, 2019-12)
      Solid organ transplantation is the preferred therapy for many patients diagnosed with end stage organ failure, however allograft rejection is a significant barrier for graft survival. Patient care involves heavy immunosuppressive drug treatment leading to elevated risk for cancer and other opportunistic infections. Hence there is a need to develop effective alternative approaches to minimize graft rejection. We focused on Human leukocyte antigen G (HLA-G), a nonclassical HLA class Ib molecule critically involved in the maintenance of maternal tolerance to semi-allogeneic fetal tissues during pregnancy and has emerged as a potential therapeutic target to control allograft rejection. We demonstrate here that the level of soluble HLA-G dimer was higher in a group of 90 patients with a functioning renal allograft compared with 40 patients who rejected (RJ) their transplants. The HLA-G dimer level was not affected by demographic status. One of the potential mechanisms in tissue organ allograft rejection involves the induction of granzymes and perforin, which are the main effector molecules expressed by CD8+ cytotoxic T lymphocytes and function to destroy allogeneic transplants. Using genomics, molecular and cellular analyses of cells from T-cell–mediated RJ and nonrejected kidney transplant patients, cells from leukocyte Ig-like receptor B1 (LILRB1) transgenic mice, humanized mice, and genetically engineered HLA-G dimer, we demonstrated a novel mechanism by which HLA-G dimer inhibits activation and cytotoxic capabilities of human CD8+ T cells. This mechanism implicated the downregulation of Granzyme B expression and the essential involvement of LILRB1. Thus, HLA-G dimer has the potential to be a specific and effective therapy for prevention of allograft rejection and prolongation of graft survival.
    • Investigating the Role of the Hdac3 Co-Repressor Complex in Glucocorticoid Signaling-Mediated Bone Marrow Lipid Storage with Age

      Pierce, Jessica Liane; Biomedical Sciences (Augusta University, 2019-12)
      Aging bone is characterized by loss of tissue density, marrow fat accumulation, and dysregulated bone marrow stromal cell (BMSC) differentiation. The contribution of the epigenetic regulator histone deactylase 3 (Hdac3) is of increasing interest in bone biology. Hdac3 expression decreases with aging, and the current model for conditional deletion of Hdac3 in Osterix-expressing osteoprogenitor cells (Hdac3-CKOOsx) exhibited an aged bone phenotype in young mice along with the novel finding of osteoblastic (Runx2+ osteogenic cells) lipid droplet storage. In addition, bone-specific loss of Hdac3 activity increases expression of the glucocorticoid (GC)-activating enzyme 11β-hydroxysteroid dehydrogenase type 1 (Hsd11b1), suggesting a mechanism for the increased lipid accumulation in aged and Hdac3-deficient BMSC-derived osteoblasts. The cofactor nuclear receptor corepressor 1 (NCoR1), which mediates Hdac3 enzymatic activity in a co-repressor complex (CRC), was proposed as a regulator of Hdac3 activity in bone. Both Hdac3 and NCoR1 expression decreased in aged osteoblasts, and the two factors exhibited synergy in downregulating the promoter activity of glucocorticoid-responsive elements. Because of the relationship between increased GC signaling and osteoporosis, the glucocorticoid receptor (GR) was investigated as a mediator of the marrow fat phenotype, with the hypothesis that loss of GR function in bone would be protective against common forms of osteoporosis. Chronic caloric restriction in WT and GR-deficient (GR-CKOOsx) mice was used as a short-term stressor to induce an osteoporotic phenotype, while aging of GR-deficient mice (where Hdac3 CRC expression naturally decreases) was used as a biologically-relevant model for dual loss of Hdac3 and the GR. Surprisingly, the loss of GR function in osteoprogenitors exacerbated bone loss and marrow fat accumulation in both models—and induced a chronic stress phenotype by increasing cellular bioenergetics and whole-body metabolic rate—providing evidence of a role for the GR in facilitating healthy bone maintenance as well as evidence for compensatory mechanisms that regulate bone biology through GC signaling. GR-deficient bone also induced changes to whole-body physiology (e.g., sarcopenia, decreased physical activity, metabolic dysfunction) that further demonstrate the intricacies of bone as an endocrine organ. The current study provides new avenues to investigate cell signaling, bioenergetics, and tissue crosstalk in osteoporotic bone.

      Atawia, Reem T.; Biomedical Sciences (Augusta University, 2019-11-05)
      An obesity epidemic continues to rise worldwide. Visceral (central) obesity is an important concern as it correlates with metabolic and cardiovascular pathologies. Arginase is a ureahydrolase enzyme with two isoforms (A1-cytosolic and A2-mitochondrial). We found that visceral adipose tissue (VAT) from obese WT mice fed a high fat/high sucrose diet (HFHS) showed a significantly higher expression of A2 compared to mice fed normal chow diet (ND). We also observed that A2 expression is upregulated 3-fold in differentiated 3T3- L1 adipocytes exposed to high levels of palmitate and glucose, a mimic of the obese state, compared to control media. Our study focused on the involvement of A2 in obesity associated metabolic and vascular disorders. WT mice and those globally lacking A2 (A2-/-) were fed HFHS or ND for 16 weeks. The HFHS diet-induced increases in body and VAT weights and total adiposity were prevented or reduced in A2-/- mice. In concert, metabolic chamber studies revealed that energy expenditure and fatty acid oxidation rates were significantly higher in A2-/- compared to WT HFHS mice. VAT from A2-/- mice fed HFHS had higher levels of active AMPK-α, the master regulator of fatty acid metabolism, as well as higher adipocyte expression of genes involved in fatty acid β-oxidation and oxidative phosphorylation, along with preserved mitochondrial density compared to WT HFHS. A2 deletion also prevented HFHS-induced fibrous tissue deposition and inflammation in VAT, which contributed to adipocyte metabolic dysfunction. These results indicate that A2 is involved in metabolic dysfunctions. To gain insights into the role of A2 in adipocytes, primary preadipocytes isolated from VAT of A2-/- mice and differentiated in vitro showed increased expression of adiponectin and better mitochondrial function. Adenoviral overexpression of A2 in differentiated 3T3-L1 cells showed impaired mitochondrial function and increased mitochondrial ROS. Obesity-related metabolic disorders increase the risk of cardiovascular diseases, the leading global cause of death. Endothelium-dependent vasorelaxation, impaired by HFHS diet, was significantly preserved in A2-/- mice, but more prominently prevented in A1+/- mice. In conclusion, A2 is critically involved in HFHS-induced obesity, VAT inflammation and metabolic dysregulation. Both A1 and A2 are involved in HFHS-induced vascular endothelial dysfunction.

      Rashid, Mohammad Harun; Department of Biochemistry and Molecular Biology (Augusta University, 2019-07)
      Exosomes are critical mediators of intercellular crosstalk and regulators of the cellular/tumor microenvironment. Exosomes have great prospects for clinical application as a theranostic and prognostic probe. Nevertheless, the advancement of exosome research has been thwarted by our limited knowledge of the most efficient isolation method and the in vivo trafficking. Here we have shown that a combination of two size-based methods using a 0.20 μm syringe filter and 100k centrifuge membrane filter followed by ultracentrifugation yields a greater number of uniform exosomes compared to other available methods. We demonstrated the visual representation and quantification of the differential in vivo distribution of radioisotope 131I-labeled exosomes from diverse cellular origins, e.g., tumor cells with or without treatments, myeloid-derived suppressor cells and endothelial progenitor cells. We also determined that the distribution was dependent on the exosomal protein/cytokine contents. Further, we also generated engineered exosomes expressing precision peptide for targeting CD206 positive M2-macrophages. M2-macrophages participate in immune suppression, epithelial to mesenchymal transition, invasion, angiogenesis, tumor progression and subsequent metastasis foci formation. Given their pro-tumorigenic function and prevalence in most malignant tumors with lower survival, early in vivo detection and intervention of M2-macrophages may boost the clinical outcome. To determine in vivo distribution of M2-macrophages, we adopted 111In-oxine based radiolabeling of the targeted exosomes and SPECT. When injected these radiolabeled targeted exosomes into 4T1 breast tumor-bearing mice, exosomes accumulated at the periphery of the primary tumor, metastatic foci in the lungs, in the spleen, and liver. Ex vivo quantification of radioactivity also showed similar distribution. Injected DiI dye-labeled exosomes into the same mice showed the adherence of exosomes to the CD206 positive macrophages on ex vivo fluorescent microscopy imaging, confirming the targeting efficacy of the exosomes. In addition, we utilized these engineered exosomes to carry the Fc portion of mouse IgG2b with the intention of augmenting antibody-dependent cell-mediated cytotoxicity. We have auspiciously demonstrated that M2-macrophage targeting therapeutic exosomes deplete M2-macrophages both in vitro and in vivo, and reduce tumor burden in a metastatic breast cancer model. The applied in vivo imaging modalities can be utilized to monitor disease progression, metastasis, and exosome-based targeted therapy.

      Ibrahim, Mohammed Mahmoud Labib; Biomedical Sciences (Augusta University, 2019-05)
      A causal link between chronic inflammation and tumorigenesis is now well established in the literature with a great deal of supporting evidences from genetic, epigenetic, pharmacological and epidemiological perspectives. In particular, inflammatory bowel diseases represent an important risk factor for colon cancer development. Moreover, it seems that even sporadic colon cancers that do not develop as a complication of chronic colitis are also driven by inflammation. However, the molecular mechanisms behind inflammation-mediated colon tumorigenesis have remained largely unknown. Colitis associated cancer development is thought to be multifaceted due to a combination of genetic and epigenetic aberrations. Recently, epigenetic alterations -particularly aberrant DNA methylation- have gained great attention in cancer biology and have been observed to play a key role in the pathogenesis of inflammation-associated tumors; especially in colitis-associated cancer. IRF8, a key transcription factor originally identified in myeloid cells, has been reported to play a crucial role in myeloid cells differentiation and immune response regulation. IRF8 deficiency is associated with deregulation of myeloid cell differentiation and accumulation of immature myeloid subsets phenotypically and functionally resemble MDSCs (Myeloid Derived Suppressor Cells). IRF8 is thought to function as a tumor suppressor and was found to be silenced in different types of cancers including colon cancer. Myeloid derived-IRF8 has been extensively studied. However, the role of epithelial-derived IRF8 in colon inflammation and colon cancer initiation remains a point to be addressed. In this study, we generated conditional Irf8cKO mice in which IRF8 is specifically deleted in colon epithelium. Irf8cKO mice exhibit a more aggressive pattern of colitis associated cancer with higher tumor incidence and severe loss of body weight. Additionally, we provide evidence that chronic inflammation promotes the accumulation and infiltration of CD11b+Gr1+ MDSCs, which plentifully secrete IL10 in colon tissue. IL10 then induces STAT3 phosphorylation and nuclear translocation to bind to Dnmt1 and Dnmt3b promoters to upregulate their expression, leading to DNA hyper-methylation at the Irf8 promoter to silence IRF8 expression in colonic epithelial cells and promote colon tumorigenesis. Collectively, our data pinpoint the MDSC-IL10-STAT3-DNMT3b-IRF8 axis as a novel bridge between chronic inflammation and colon cancer formation.