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The Effect of Immunoproteasome Inhibition on T Helper Cells in Acute Inflammation

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The immunoproteasome is a variant of the 26S proteasome in which the catalytically active standard β-subunits (β1c, β2c and β5c) are replaced by low molecular mass polypeptide (LMP)2 (β1i), multicatalytic endopeptidase complex-like (MECL)-1 (β2i) and LMP7 (β5i). Besides its role in antigen presentation, many studies have shown that the immunoproteasome is important in activation and differentiation of T helper (Th) cells, cytokine secretion and development of autoimmune disorders, making it an interesting therapeutic target for T cell-mediated diseases. Indeed, irreversible inhibition of the immunoproteasome subunits LMP2/LMP7 by ONX 0914 has shown great effects in ameliorating disease symptoms in various mouse models of inflammatory diseases and in reducing the frequency of IFN-γ+ Th1 and IL-17+ Th17 cells. In contrast, little is known about the role of the immunoproteasome in the development of allergic Th2-mediated inflammation. Allergic asthma is induced by inhalation of environmental allergens which trigger an excessive immune response, resulting in high levels of inflammatory cytokines like IL-4, IL-5 and IL-13, eosinophilic infiltration and increased immunoglobulin E (IgE) production by B cells. Antigen-specific Th2 cells are central mediators of this allergic reaction. Even though most patients benefit from the standard corticosteroid treatment, a large proportion of patients develops corticosteroid-resistant asthma, leading to a great need of novel therapeutics. The first part of this study aimed to investigate the role of the immunoproteasome in CD4+ Th cells. In the first set of experiments, the focus was Th2 cells and the therapeutic potential of immunoproteasome inhibition on Th2 cells in allergic airway inflammation. First, it was shown that in vitro polarized Th cell subsets (Th1, Th2, Th17, and Treg) express high levels of immunoproteasome. For in vivo analyses allergic airway inflammation was induced in mice of the novel GATA-3-reporter strain GATIR which are useful for easy identification of Th2 cells. ONX 0914 was applied during the challenge phase to investigate the influence on the acute Th2 response. Furthermore, the development of Th2 cells was analyzed under the influence of ONX 0914 which was applied continuously starting from the first sensitization. When ONX 0914 was applied during the challenge phase, a reduction of the Th2 response was detected in two different models, using ovalbumin or house dust mite (HDM) extract as allergens. Furthermore, T cell activation and antigen-specific cytokine secretion was impaired and a reduced infiltration of eosinophils and professional antigen presenting cells into the lung and the bronchoalveolar space was observed in the ovalbumin model. In contrast, there was no difference in the Th2 response or airway inflammation, when ONX 0914 was applied starting from the first sensitization. Moreover, the development of ovalbumin-induced airway inflammation was similar in wildtype and LMP7-deficient mice. In both cases this coincided with increased expression of the standard subunit β5c, suggesting a compensatory mechanism. Taken together, it could be shown that primed Th2 cells are sensitive to III immunoproteasome inhibition and that allergic airway inflammation could be reduced but early ONX 0914 treatment or genetic deficiency of LMP7 could not prevent disease development. Even though many studies have demonstrated that Th17 cells are diminished upon immunoproteasome inhibition, it is still unclear if differentiation or survival of these cells is affected. In a second line of experiments, dextran sodium sulfate (DSS) was therefore used to induce colitis in mice and to investigate the effect of immunoproteasome inhibition on Th17 cells at different time points, i.e. during differentiation and later on established Th17 cells. In the same manner, Tregs were examined as previous studies yielded opposing data about the immunoproteasome in Tregs. In line with previous studies, the frequency of Th17 cells in the lamina propria of the colon was almost abolished when ONX 0914 was applied continuously during DSS colitis. In contrast, ONX 0914 did not decrease already induced Th17 cells at a later time point. In both settings, Tregs in the lamina propria were not affected by ONX 0914 treatment. In conclusion, different Th cell subsets require immunoproteasome function at distinct time points and respond differently to ONX 0914. In our setup, Th2 cells could develop independently of immunoproteasome function but Th17 cells required it. In contrast, primed Th2 cells but not established Th17 cells were affected by immunoproteasome inhibition. Comparative studies of degradation characteristics of the standard- and immunoproteasome are often performed with isolated proteasomes in vitro. Since this might not reflect the real physiologic situation in cellulo, the aim of the second part of this thesis was to establish a stable cell line for inducible immunoproteasome expression. To our knowledge, the only currently available comparable cell line is a tetracycline-inducible murine fibroblast line. Since fibroblasts normally do not express immunoproteasomes, the aim was to establish a line based on cells usually expressing almost exclusively immunoproteasomes. To this end, the immortalized B cell line LCL721.174, lacking the genomic region where the genes encoding for LMP2 and LMP7 are located, was used as a basis for a tamoxifen-inducible system. Three stable clones with robust expression after 7 days of tamoxifen treatment were established. This cell line was then used for proteomics analysis (SILAC - stable isotope labeling with amino acids in cell culture) to identify potential substrates specific for one of the two proteasome types. The best hit was a reduction of nuclear factor NF-κB in immunoproteasome samples. Cycloheximide chase experiments were performed to confirm this but no differences could be detected. Furthermore, this cell line was used to investigate the degradation of poly-ubiquitinated proteins and the susceptibility to oxidative stress, but no differences were detected between the two samples.

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ISO 690OLIVERI, Franziska, 2022. The Effect of Immunoproteasome Inhibition on T Helper Cells in Acute Inflammation [Dissertation]. Konstanz: University of Konstanz
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@phdthesis{Oliveri2022Effec-59544,
  year={2022},
  title={The Effect of Immunoproteasome Inhibition on T Helper Cells in Acute Inflammation},
  author={Oliveri, Franziska},
  address={Konstanz},
  school={Universität Konstanz}
}
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    <dcterms:abstract xml:lang="eng">The immunoproteasome is a variant of the 26S proteasome in which the catalytically active standard β-subunits (β1c, β2c and β5c) are replaced by low molecular mass polypeptide (LMP)2 (β1i), multicatalytic endopeptidase complex-like (MECL)-1 (β2i) and LMP7 (β5i). Besides its role in antigen presentation, many studies have shown that the immunoproteasome is important in activation and differentiation of T helper (Th) cells, cytokine secretion and development of autoimmune disorders, making it an interesting therapeutic target for T cell-mediated diseases. Indeed, irreversible inhibition of the immunoproteasome subunits LMP2/LMP7 by ONX 0914 has shown great effects in ameliorating disease symptoms in various mouse models of inflammatory diseases and in reducing the frequency of IFN-γ+ Th1 and IL-17+ Th17 cells. In contrast, little is known about the role of the immunoproteasome in the development of allergic Th2-mediated inflammation. Allergic asthma is induced by inhalation of environmental allergens which trigger an excessive immune response, resulting in high levels of inflammatory cytokines like IL-4, IL-5 and IL-13, eosinophilic infiltration and increased immunoglobulin E (IgE) production by B cells. Antigen-specific Th2 cells are central mediators of this allergic reaction. Even though most patients benefit from the standard corticosteroid treatment, a large proportion of patients develops corticosteroid-resistant asthma, leading to a great need of novel therapeutics. The first part of this study aimed to investigate the role of the immunoproteasome in CD4+ Th cells. In the first set of experiments, the focus was Th2 cells and the therapeutic potential of immunoproteasome inhibition on Th2 cells in allergic airway inflammation. First, it was shown that in vitro polarized Th cell subsets (Th1, Th2, Th17, and Treg) express high levels of immunoproteasome. For in vivo analyses allergic airway inflammation was induced in mice of the novel GATA-3-reporter strain GATIR which are useful for easy identification of Th2 cells. ONX 0914 was applied during the challenge phase to investigate the influence on the acute Th2 response. Furthermore, the development of Th2 cells was analyzed under the influence of ONX 0914 which was applied continuously starting from the first sensitization. When ONX 0914 was applied during the challenge phase, a reduction of the Th2 response was detected in two different models, using ovalbumin or house dust mite (HDM) extract as allergens. Furthermore, T cell activation and antigen-specific cytokine secretion was impaired and a reduced infiltration of eosinophils and professional antigen presenting cells into the lung and the bronchoalveolar space was observed in the ovalbumin model. In contrast, there was no difference in the Th2 response or airway inflammation, when ONX 0914 was applied starting from the first sensitization. Moreover, the development of ovalbumin-induced airway inflammation was similar in wildtype and LMP7-deficient mice. In both cases this coincided with increased expression of the standard subunit β5c, suggesting a compensatory mechanism. Taken together, it could be shown that primed Th2 cells are sensitive to III immunoproteasome inhibition and that allergic airway inflammation could be reduced but early ONX 0914 treatment or genetic deficiency of LMP7 could not prevent disease development. Even though many studies have demonstrated that Th17 cells are diminished upon immunoproteasome inhibition, it is still unclear if differentiation or survival of these cells is affected. In a second line of experiments, dextran sodium sulfate (DSS) was therefore used to induce colitis in mice and to investigate the effect of immunoproteasome inhibition on Th17 cells at different time points, i.e. during differentiation and later on established Th17 cells. In the same manner, Tregs were examined as previous studies yielded opposing data about the immunoproteasome in Tregs. In line with previous studies, the frequency of Th17 cells in the lamina propria of the colon was almost abolished when ONX 0914 was applied continuously during DSS colitis. In contrast, ONX 0914 did not decrease already induced Th17 cells at a later time point. In both settings, Tregs in the lamina propria were not affected by ONX 0914 treatment. In conclusion, different Th cell subsets require immunoproteasome function at distinct time points and respond differently to ONX 0914. In our setup, Th2 cells could develop independently of immunoproteasome function but Th17 cells required it. In contrast, primed Th2 cells but not established Th17 cells were affected by immunoproteasome inhibition. Comparative studies of degradation characteristics of the standard- and immunoproteasome are often performed with isolated proteasomes in vitro. Since this might not reflect the real physiologic situation in cellulo, the aim of the second part of this thesis was to establish a stable cell line for inducible immunoproteasome expression. To our knowledge, the only currently available comparable cell line is a tetracycline-inducible murine fibroblast line. Since fibroblasts normally do not express immunoproteasomes, the aim was to establish a line based on cells usually expressing almost exclusively immunoproteasomes. To this end, the immortalized B cell line LCL721.174, lacking the genomic region where the genes encoding for LMP2 and LMP7 are located, was used as a basis for a tamoxifen-inducible system. Three stable clones with robust expression after 7 days of tamoxifen treatment were established. This cell line was then used for proteomics analysis (SILAC - stable isotope labeling with amino acids in cell culture) to identify potential substrates specific for one of the two proteasome types. The best hit was a reduction of nuclear factor NF-κB in immunoproteasome samples. Cycloheximide chase experiments were performed to confirm this but no differences could be detected. Furthermore, this cell line was used to investigate the degradation of poly-ubiquitinated proteins and the susceptibility to oxidative stress, but no differences were detected between the two samples.</dcterms:abstract>
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December 9, 2022
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Konstanz, Univ., Diss., 2022
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