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Targeting the immunoproteasome and VCP/p97 in autoimmune disorders and viral infection

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2022

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The proteasome is a multicatalytic system involved in the degradation of polyubiquitinated proteins. Thus, the proteasome is key to maintaining cell homeostasis. Stress and pro-inflammatory cytokines prompt the replacement of the proteasome catalytic subunits β1c, β2c, and β5c by the inducible subunits low molecular mass polypeptide (LMP)2, multicatalytic endopeptidase complex-like (MECL)-1, and LMP7. Since the expression of immunoproteasome is restricted to cells of hematopoietic origin and sites of inflammation, targeting the immunoproteasome overcomes the toxicities associated with proteasome inhibitors. Indeed, immunoproteasome inhibitors were shown to ameliorate the clinical symptoms in murine models of autoimmune disorders such as rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, and diabetes. The immunoproteasome is widely known for shaping the antigenic repertoire and controlling the antigen presentation on major histocompatibility complex (MHC) class I molecules. However, it also plays a role in cytokine production and T helper (Th) cell differentiation by suppressing the development of Th1 and Th17 cells. Due to the critical roles of the immunoproteasome modulating the immune response, we addressed the question of whether selective inhibition of LMP7 could be used as a therapeutic approach to treat polymyositis (PM) and psoriasis. PM is an idiopathic inflammatory myopathy (IIM) that causes proximal muscle weakness. The current lack of specific treatments highlights the difficulties in disease management. In our study, we use a murine model of PM designated C-protein included myositis (CIM) that mimics PM by injecting a recombinant fragment of the myosin-binding protein C2. Using two immunoproteasome inhibitors, ONX 0914 and KZR-616, we demonstrated that immunoproteasome inhibition normalized the declined grip strength associated with PM while reducing histopathological lesions in the skeletal muscle and the creatine kinase levels in the serum. Characterization of the inflammatory infiltrate in the muscle showed that CD8+ and F4/80+ cells were almost absent in the tissue after treatment. Furthermore, we observed that LMP7-/- mice were resistant to CIM induction. On the other hand, psoriasis is an autoimmune disorder characterized by the formation of red plaques. Current treatments are usually discontinued and lead to refractory disease. In this study, we demonstrate that ONX 0914 ameliorated the psoriasis-like symptoms inCard14ΔE138+/- mice and imiquimod-induced psoriasiform murine model in a therapeutic setup. Administration of ONX 0914 led to a reduction of the ear thickness and the pathological lesions in the tissue. In addition, flow cytometry and immunofluorescence studies demonstrated a reduction in the inflammatory infiltrate in both models. Moreover, several pro-inflammatory cytokines associated with psoriasis pathogenesis were normalized in the tissue. In the Card14-mediated murine model, we observed a switch in the αβ+ and γδ+ populations and their corresponding IL-17A and IL-22 secretion after treatment with ONX 0914. In contrast, in the imiquimod-murine model, we observed the normalization of the IL-17A levels in the serum after treatment with the immunoproteasome inhibitor. This data demonstrates immunoproteasome inhibitors' therapeutic benefits in two murine models of psoriasis. Finally, we focused on the valosin-containing protein (VCP/p97), an upstream element of the proteasome that enables the extraction of peptides from protein complexes, allowing them to be proteolytically processed by the proteasome. Due to the crucial role of VCP/p97 in cell homeostasis and the wide range of cellular functions attributed to this ATPase, it is not surprising that many viruses exploit this element. Indeed, little is known about the role of VCP/p97 during in vivo infection. To unveil this topic, we administered CB-5083, an orally bioavailable inhibitor of VCP/p97, during lymphocytic choriomeningitis virus (LCMV) infection. We observed that LCMV infection increased the susceptibility to CB-5083, causing an alteration in the viral clearance in a CD8+-independent manner. Furthermore, the administration of CB-5083 during LCMV infection affected several immune populations in the blood and the spleen. We also observed that LCMV infection upregulated both ATF4 and XBP1s branches of the unfolded protein response in vitro, while the addition of CB-5083 led to the upregulation of the XBP1 branch in a time-dependent manner. In contrast, administration of CB-5083 for 6 hours after the priming phase of the lymphocytes led to massive toxicity and the accumulation of polyubiquitinated proteins in the spleen. In addition, the analysis of protein aggresomes in several tissues demonstrated that aggresomes explicitly accumulated in the skeletal muscle after treatment with CB-5083. Moreover, the creatine kinase and lactate dehydrogenase were upregulated in the serum of the treated mice, demonstrating that inhibition of VCP/p97 during the late phase of LCMV infection led to toxicity related to muscular damage. Taken together, the studies described in this thesis suggest that immunoproteasome inhibition can be a therapeutic target in autoimmune disorders, specifically PM and psoriasis. Furthermore, we assessed the toxicities associated with the in vivo administration of VCP/97 inhibitors and their association with muscular damage.

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570 Biowissenschaften, Biologie

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ISO 690DEL RIO OLIVA, Marta, 2022. Targeting the immunoproteasome and VCP/p97 in autoimmune disorders and viral infection [Dissertation]. Konstanz: University of Konstanz
BibTex
@phdthesis{DelRioOliva2022Targe-59543,
  year={2022},
  title={Targeting the immunoproteasome and VCP/p97 in autoimmune disorders and viral infection},
  author={Del Rio Oliva, Marta},
  address={Konstanz},
  school={Universität Konstanz}
}
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The immunoproteasome is widely known for shaping the antigenic repertoire and controlling the antigen presentation on major histocompatibility complex (MHC) class I molecules. However, it also plays a role in cytokine production and T helper (Th) cell differentiation by suppressing the development of Th1 and Th17 cells. Due to the critical roles of the immunoproteasome modulating the immune response, we addressed the question of whether selective inhibition of LMP7 could be used as a therapeutic approach to treat polymyositis (PM) and psoriasis. PM is an idiopathic inflammatory myopathy (IIM) that causes proximal muscle weakness. The current lack of specific treatments highlights the difficulties in disease management. In our study, we use a murine model of PM designated C-protein included myositis (CIM) that mimics PM by injecting a recombinant fragment of the myosin-binding protein C2. Using two immunoproteasome inhibitors, ONX 0914 and KZR-616, we demonstrated that immunoproteasome inhibition normalized the declined grip strength associated with PM while reducing histopathological lesions in the skeletal muscle and the creatine kinase levels in the serum. Characterization of the inflammatory infiltrate in the muscle showed that CD8+ and F4/80+ cells were almost absent in the tissue after treatment. Furthermore, we observed that LMP7-/- mice were resistant to CIM induction. On the other hand, psoriasis is an autoimmune disorder characterized by the formation of red plaques. Current treatments are usually discontinued and lead to refractory disease. In this study, we demonstrate that ONX 0914 ameliorated the psoriasis-like symptoms inCard14ΔE138+/- mice and imiquimod-induced psoriasiform murine model in a therapeutic setup. Administration of ONX 0914 led to a reduction of the ear thickness and the pathological lesions in the tissue. In addition, flow cytometry and immunofluorescence studies demonstrated a reduction in the inflammatory infiltrate in both models. Moreover, several pro-inflammatory cytokines associated with psoriasis pathogenesis were normalized in the tissue. In the Card14-mediated murine model, we observed a switch in the αβ+ and γδ+ populations and their corresponding IL-17A and IL-22 secretion after treatment with ONX 0914. In contrast, in the imiquimod-murine model, we observed the normalization of the IL-17A levels in the serum after treatment with the immunoproteasome inhibitor. This data demonstrates immunoproteasome inhibitors' therapeutic benefits in two murine models of psoriasis. Finally, we focused on the valosin-containing protein (VCP/p97), an upstream element of the proteasome that enables the extraction of peptides from protein complexes, allowing them to be proteolytically processed by the proteasome. Due to the crucial role of VCP/p97 in cell homeostasis and the wide range of cellular functions attributed to this ATPase, it is not surprising that many viruses exploit this element. Indeed, little is known about the role of VCP/p97 during in vivo infection. To unveil this topic, we administered CB-5083, an orally bioavailable inhibitor of VCP/p97, during lymphocytic choriomeningitis virus (LCMV) infection. We observed that LCMV infection increased the susceptibility to CB-5083, causing an alteration in the viral clearance in a CD8+-independent manner. Furthermore, the administration of CB-5083 during LCMV infection affected several immune populations in the blood and the spleen. We also observed that LCMV infection upregulated both ATF4 and XBP1s branches of the unfolded protein response in vitro, while the addition of CB-5083 led to the upregulation of the XBP1 branch in a time-dependent manner. In contrast, administration of CB-5083 for 6 hours after the priming phase of the lymphocytes led to massive toxicity and the accumulation of polyubiquitinated proteins in the spleen. In addition, the analysis of protein aggresomes in several tissues demonstrated that aggresomes explicitly accumulated in the skeletal muscle after treatment with CB-5083. Moreover, the creatine kinase and lactate dehydrogenase were upregulated in the serum of the treated mice, demonstrating that inhibition of VCP/p97 during the late phase of LCMV infection led to toxicity related to muscular damage. Taken together, the studies described in this thesis suggest that immunoproteasome inhibition can be a therapeutic target in autoimmune disorders, specifically PM and psoriasis. Furthermore, we assessed the toxicities associated with the in vivo administration of VCP/97 inhibitors and their association with muscular damage.</dcterms:abstract>
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December 6, 2022
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Konstanz, Univ., Diss., 2022
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