Ahmed S. Elshikha

Alpha 1 Antitrypsin Inhibits Dendritic Cell Activation and Attenuates Nephritis in a Mouse Model of Lupus

Systemic lupus erythematosus (SLE) is an autoimmune disorder with a worldwide distribution and considerable mortality and morbidity. Although the pathogenesis of this disease remains elusive, over-reactive dendritic cells (DCs) play a critical role in the disease development. It has been shown that human alpha-1 antitrypsin (hAAT) has protective effects in type 1 diabetes and rheumatoid arthritis mouse models. In the present study, we tested the effect of AAT on DC differentiation and functions, as well as its protective effect in a lupus-prone mouse model. We showed that hAAT treatment significantly inhibited LPS (TLR4 agonist) and CpG (TLR9 agonist) -induced bone-marrow (BM)-derived conventional and plasmacytoid DC (cDC and pDC) activation and reduced the production of inflammatory cytokines including IFN-I, TNF-α and IL-1β. In MRL/lpr mice, hAAT treatment significantly reduced BM-derived DC differentiation, serum autoantibody levels, and importantly attenuated renal pathology. Our results for the first time demonstrate that hAAT inhibits DC activation and function, and it also attenuates autoimmunity and renal damage in the MRL/lpr lupus model. These results imply that hAAT has a therapeutic potential for the treatment of SLE in humans.

In Situ Transplantation of Alginate Bioencapsulated Adipose Tissues Derived Stem Cells (ADSCs) via Hepatic Injection in a Mouse Model

Objective Adipose tissue derived stem cells (ADSCs) transplantation has recently gained widespread enthusiasm, particularly in the perspective to use them as potential alternative cell sources for hepatocytes in cell based therapy, mainly because of their capability of hepatogenic differentiation in vitro and in vivo. But some challenges remain to be addressed, including whether ADSCs can be provided effectively to the target organ and whether subsequent proliferation of transplanted cells can be achieved. To date, intrasplenic injection is the conventional method to deliver ADSCs into the liver; however, a number of donor cells retained in the spleen has been reported. In this study, our objective is to evaluate a novel route to transplant ADSCs specifically to the liver. We aimed to test the feasibility of in situ transplantation of ADSCs by injecting bioencapsulated ADSCs into the liver in mouse model. Methods The ADSCs isolated from human alpha 1 antitrypsin (M-hAAT) transgenic mice were used to allow delivered ADSCs be readily identified in the liver of recipient mice, and alginate was selected as a cell carrier. We first evaluated whether alginate microspheres are implantable into the liver tissue by injection and whether ADSCs could migrate from alginate microspheres (study one). Once proven, we then examined the in vivo fate of ADSCs loaded microspheres in the liver. Specifically, we evaluated whether transplanted, undifferentiated ASDCs could be induced by the local microenvironment toward hepatogenic differentiation and the distribution of surviving ADSCs in major tissue organs (study two). Results Our results indicated ADSCs loaded alginate microspheres were implantable into the liver. Both degraded and residual alginate microspheres were observed in the liver up to three weeks. The viable ADSCs were detectable surrounding degraded and residual alginate microspheres in the liver and other major organs such as bone marrow and the lungs. Importantly, transplanted ADSCs underwent hepatogenic differentiation to become cells expressing albumin in the liver. These findings improve our understanding of the interplay between ADSCs (donor cells), alginate (biomaterial), and local microenvironment in a hepatectomized mouse model, and might improve the strategy of in situ transplantation of ADSCs in treating liver diseases.

In Vivo Analysis of Alpha-1-Antitrypsin Functions in Autoimmune Disease Models

Alpha-1-antitrypsin (AAT) is a circulating protein, a serpin, with multiple protective functions. Beside the well-known proteinase inhibitory function, which protects the lungs from chronic obstructive pulmonary disease (COPD), many studies have shown that AAT inhibits pro-inflammatory cytokine gene expression and functions. These anti-inflammatory and immune-regulatory properties have led to studies testing the therapeutic effect of AAT in autoimmune disease models. Initially, a study using recombinant adeno-associated viral (rAAV) vector showed that AAT gene therapy prevented type 1 diabetes (T1D) development in a nonobese diabetic (NOD) mouse model. Consequently, several studies confirmed that AAT therapy prevented and reversed T1D. AAT therapy has also been tested and has demonstrated protective effects in a collagen-induced arthritis model and a systemic lupus erythematosus (SLE) mouse model. This chapter describes methods that evaluate AAT functions in autoimmune mouse models.

Alpha 1 antitrypsin gene therapy extends the life-span of lupus-prone mice

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease characterized by high levels of pathogenic autoantibodies and tissue damage. Multiple studies showed that dendritic cell (DC) activation plays a critical role in SLE pathogenesis. Human alpha 1 antitrypsin (hAAT) is a serine proteinase inhibitor with potent anti-inflammatory and cytoprotective properties. In this study, we first examined the effects of hAAT on the functions of DCs from lupus-prone mice, and we showed that hAAT treatment efficiently inhibited CpG- (TLR9 agonist) induced activation of bone marrow-derived conventional and plasmacytoid DCs as well as the production of pro-inflammatory cytokines. The hAAT treatment also attenuated DC help for B cell proliferation and immunoglobulin M (IgM) production. We next tested the protective effect of hAAT protein and gene therapy using recombinant adeno-associated virus 8 (rAAV8-CB-hAAT) in a spontaneous lupus mouse model, and we showed that both treatments decreased autoantibody levels. Importantly, rAAV8-CB-hAAT did not induce an immune response to its transgene product (hAAT), but it showed more pronounced therapeutic effects in reducing urine protein levels and extending the lifespan of these mice. These results indicate that AAT has therapeutic potential in the treatment of SLE in humans.

Anti-inflammaging effects of human alpha-1 antitrypsin

Inflammaging plays an important role in most age‐related diseases. However, the mechanism of inflammaging is largely unknown, and therapeutic control of inflammaging is challenging. Human alpha‐1 antitrypsin (hAAT) has immune‐regulatory, anti‐inflammatory, and cytoprotective properties as demonstrated in several disease models including type 1 diabetes, arthritis, lupus, osteoporosis, and stroke. To test the potential anti‐inflammaging effect of hAAT, we generated transgenic Drosophila lines expressing hAAT. Surprisingly, the lifespan of hAAT‐expressing lines was significantly longer than that of genetically matched controls. To understand the mechanism underlying the anti‐aging effect of hAAT, we monitored the expression of aging‐associated genes and found that aging‐induced expressions of Relish (NF‐ĸB orthologue) and Diptericin were significantly lower in hAAT lines than in control lines. RNA‐seq analysis revealed that innate immunity genes regulated by NF‐kB were significantly and specifically inhibited in hAAT transgenic Drosophila lines. To confirm this anti‐inflammaging effect in human cells, we treated X‐ray‐induced senescence cells with hAAT and showed that hAAT treatment significantly decreased the expression and maturation of IL‐6 and IL‐8, two major factors of senescence‐associated secretory phenotype. Consistent with results from Drosophila,RNA‐seq analysis also showed that hAAT treatment significantly inhibited inflammation related genes and pathways. Together, our results demonstrated that hAAT significantly inhibited inflammaging in both Drosophila and human cell models. As hAAT is a FDA‐approved drug with a confirmed safety profile, this novel therapeutic potential may make hAAT a promising candidate to combat aging and aging‐related diseases.

84. Alpha 1 Antitrypsin Protein & Gene Therapies for the Treatment of Lupus in Animal Models

Systemic lupus erythematosus (SLE) is an autoimmune rheumatic disorder. Although the pathogenesis of SLE remains elusive, over-reactive dendritic cells (DCs), which promote B cell activation and autoantibodies secretion, play critical roles in the disease development. Alpha-1 antitrypsin (AAT) is a multifunctional protein with anti-inflammatory, cytoprotective and immunoregulatory properties. We has shown that AAT has protective effect in autoimmune disease models including type 1 diabetes and rheumatoid arthritis. In this study, we tested therapeutic potentials of AAT in controlling DC and B cells function and the development of SLE in a spontaneous lupus mouse model. Using bone marrow cells from normal (C57BL/6) and lupus prone (B6. TC, MRL/lpr) mice, we showed that AAT treatment significantly inhibited lipopolysaccharide (LPS) (TLR4 agonist) or CpG (TLR9 agonist) induced DC (cDC and pDC) maturation. In this in vitro system, AAT significantly inhibited the production of IFN-I, IL-1β, IL-6 and TNF-α from DCs. AAT treated DC also have significantly lower potential in stimulating B cell proliferation and functions. Based on these results, performed following experiments using spontaneous lupus mouse models. In order to test this therapeutic potential, we treated female MRL/lpr mice (at 7-weeks of age, n=10) with clinical grade of AAT (2 mg/mouse, every 3 days) or PBS for 11 weeks. Consistent with our in vitro observations, results from this experiment showed that AAT treatment significantly inhibited DC maturation and reduced DC susceptibility to LPS stimulation. Importantly, AAT treatment significantly lowered serum antibody (anti-dsDNA IgG and anti-nuclear antibodies, ANAs) levels and urine albumin levels. Detailed pathological examinations showed that AAT treatment prevented kidney disease development. To confirm this observations and test the long-term effect of AAT treatment, we performed second experiment using both AAT protein and gene therapies. In this experiment, we treated adult female NZM2410 (another commonly used spontaneous lupus model) mice with AAT protein and rAAV8-CB-AAT vector (1×1011 vg/mouse, single IP injection). While 100% mice in control group develop lupus and died at 46 week of age, 50% mice in AAT protein treatment group remain lupus free at 55 weeks of age. Intriguingly, AAT gene therapy (rAAV8-CB-AAT) significantly prevent lupus development (70% mice remain lupus free at 63 weeks of age, P=0.0059). These results clearly demonstrated the therapeutic effect of AAT protein and gene therapy in lupus mouse model. Our results also indicated the advantage of rAAV8 mediated gene therapy for the chronic autoimmune diseases. In summary, we showed AAT inhibited the activation and function of DCs and B cells in vitro and in vivo. We also showed AAT treatment (protein therapy and gene therapy) prevent lupus development in spontaneous lupus mouse models. Considering the safety profile of AAT and rAAV, our results may be translated into clinical application and lead to a novel (safe and effective) therapy for SLE in humans. This work was supported by grants from University of Florida and Grifols, Inc.

632. Alpha-1 Antitrypsin Gene Therapy Prevented Bone Loss in an Ovariectomy Induced Osteoporosis Mouse Model

Osteoporosis is a major healthcare burden affecting mostly postmenopausal women characterized by compromised bone strength and increased risk of fragility fracture. Although pathogenesis of this disease is complex, inflammation is clearly involved in bone loss at menopause. Therefore, anti-inflammatory strategies hold great potential for the prevention of postmenopausal osteoporosis. Human alpha-1 antitrypsin (hAAT) is a multifunctional protein that has anti-inflammatory and cytoprotective properties. In this study, we investigated the protective effect of hAAT against bone loss. In vitro studies showed that hAAT significantly inhibited osteoclast formation and function in a dose-dependent manner. Treatment of hAAT inhibited M-CSF (macrophage colony-stimulating factor) induced cell surface RANK receptor expression by downregulating cFos mRNA expression. To test the protective effect of hAAT in an osteoporosis mouse model, we treated ovariectomized (OVX) mice with rAAV8-CB-hAAT, or mesenchymal stem cells (MSCs) infected with a lentiviral vector expressing hAAT (MSC-Lenti-hAAT) or phosphate buffer saline (PBS). Sham operated age-matched animals were used as controls. Eight weeks after the treatment, animals were sacrificed and subjected to µCT scanning for the evaluation of vertebral bone microarchitecture. Gene and stem cell-based hAAT therapies significantly increased bone volume density, trabecular number and decreased structure model index compared to PBS injection in OVX mice. Gene therapy also increased connectivity, density and trabecular thickness compared to PBS injection in OVX mice. We also observed that both therapies inhibited RANK gene expression in bone, which is consistent with the results of our in vitro study. These results demonstrate that hAAT gene and MSCs based therapies mitigate ovariectomy-induced bone loss in a mouse model, possibly through inhibition of osteoclast formation by reducing RANK gene expression. Considering the safety profile of the hAAT and rAAV vector in human, our results provide a new insight for the treatment of osteoporosis.

508. Alpha-1 Antitrypsin (AAT) Gene and Stem Cell Based Therapies for the Treatment of Osteoporosis

Osteoporosis, a serious public health problem affecting millions of Americans, is characterized by reduced bone mass and poor bone microarchitecture which can result in fractures. Although the etiology and pathogenesis of osteoporosis are complex and multifactorial, inflammation is clearly involved in the disease development. Therefore, anti-inflammatory strategy holds great potential for the prevention and treatment of this disease. Alpha 1 antitrypsin (AAT) is a multifunctional protein with anti-inflammatory, proteinase inhibitory and cytoprotective properties. We previously showed that AAT therapy had therapeutic effect in inflammation related disease models including type 1 diabetes, rheumatoid arthritis and stroke. In this study, we tested the effect of AAT on osteoclastogenesis and the therapeutic potential of AAT for preventing bone loss in an ovariectomized (OVX) mouse model. In vitro studies showed that AAT significantly inhibited osteoclast formation in a dose-dependent manner. AAT also inhibited the gene expressions of TNF-alpha and receptor activator of nuclear factor kappa B (RANK) in the early and late stages of osteoclast differentiation. To test the protective effect of AAT on osteoporosis, OVX mice were injected with either clinical grade AAT (2 mg/mouse/3 days), recombinant adeno associated virus vector expressing AAT (rAAV8-CB-AAT, 1×1011 particles/mouse), mesenchymal stem cells (MSCs) infected with lentiviral vector expressing AAT (MSC-Lenti-AAT) or PBS (100 microliter/mouse/3 days). We used age-matched and sham operated animals as a normal control. Eight weeks after the treatment, all animals were sacrificed and subjected to μCT scanning for the evaluation of vertebra bone microarchitecture including trabecular BV (bone volume), TV (Total volume), BV/TV (bone volume density), Tb.N (trabecular number), Tb.Th (trabecular thickness), Conn.Dn (connectivity density), Tb.Sp (trabecular separation) and SMI (structure model index). Results from this study showed that AAT therapies (protein, gene and stem cell-based) significantly increased TV/BV and Tb.N, and decreased SMI compared to PBS injection in OVX mice. Gene therapy also increased Conn.Dn and Tb.Th compared to PBS injection in OVX mice. These results demonstrate that AAT protein, gene and MSCs based therapies mitigate ovariectomy-induced bone loss in mouse model. Since AAT protein and gene therapies have been proven to be safe in humans, our results not only demonstrate novel functions of AAT, but also imply a new strategy for the treatment of osteoporosis.

98. Bortezomib Enhances AAV Vector Mediated Transduction But Inhibits the Secretion of Transgene Product

Adeno-associated virus (rAAV) mediated gene therapy has shown promise for the treatment of alpha1 antitrypsin deficiency (AATD). Sustained alpha 1 antitrypsin (AAT) expression has been achieved in clinical trails, but the serum AAT concentrations do not reach therapeutic levels. Enhancing transduction efficiency is critical to developing a successful treatment. Since rAAV transduction may be limited by the proteasome, which degrade AAV capsids, we tested the effect of proteasome inhibitors on rAAV mediated AAT production. We treated HeLa and 293 cells with proteasome inhibitors including bortezomib and infected HeLa cells with rAAV2-GFP, rAAV2-AAT or AAV1-AAT and we Infected 293 cells with rAAV2-AAT. We measured AAT in the medium and the lysates of cells. Consistent with previous reports, bortezomib significantly enhanced AAV2 mediated GFP transduction in Hela cells. In rAAV-AAT infected cells, bortezomib treatment resulted in 9.7-13.1 fold increase in of intracellular AAT, while only modest (1.16-1.6 fold) increases of secreted AAT compared to that in cells without bortezomib. Similarly, intramuscular injection of rAAV1-AAT to C57BL6 mice treated with bortezomib also resulted in a small (~1 fold) increases in serum AAT compared with no treatment. These results indicate that bortezomib inhibited AAT protein secretion. To test the role unfolded protein response (UPR) in AAT accumulation, we measured UPR-related genes and showed that bortezomib treatment increased the mRNA levels of the protein chaperone and UPR sensor GRP78 (BiP) indicating an activation of ER stress pathways. Together, our results indicate that bortezomib can enhance rAAV transduction, but it can also inhibit the secretion of transgene product, which will limit the application of this drug for enhancing the efficiency of certain gene therapy, such as alpha 1 antitrypsin gene therapy.