Co-inhibition of immunoproteasome subunits LMP2 and LMP7 is required to block autoimmunity
2018-12-05, Basler, Michael, Lindstrom, Michelle M., LaStant, Jacob J., Bradshaw, J. Michael, Owens, Timothy D., Schmidt, Christian, Maurits, Elmer, Tsu, Christopher, Overkleeft, Herman S., Gröttrup, Marcus
Cells of hematopoietic origin express high levels of the immunoproteasome, a cytokine-inducible proteasome variant comprising the proteolytic subunits LMP2 (β1i), MECL-1 (β2i), and LMP7 (β5i). Targeting the immunoproteasome in pre-clinical models of autoimmune diseases with the epoxyketone inhibitor ONX 0914 has proven to be effective. ONX 0914 was previously described as a selective LMP7 inhibitor. Here, we show that PRN1126, developed as an exclusively LMP7-specific inhibitor, has limited effects on IL-6 secretion, experimental colitis, and experimental autoimmune encephalomyelitis (EAE). We demonstrate that prolonged exposure of cells with ONX 0914 leads to inhibition of both LMP7 and LMP2. Co-inhibition of LMP7 and LMP2 with PRN1126 and LMP2 inhibitors LU-001i or ML604440 impairs MHC class I cell surface expression, IL-6 secretion, and differentiation of naïve T helper cells to T helper 17 cells, and strongly ameliorates disease in experimental colitis and EAE. Hence, co-inhibition of LMP2 and LMP7 appears to be synergistic and advantageous for the treatment of autoimmune diseases.
Multi-THz nonlinear optics and sub-cycle control of charge and spin
2013, Porer, Michael, Sell, Alexander, Junginger, Friederike, Mayer, Bernhard, Schmidt, Christian, Mährlein, Sebastian, Pashkin, Alexej, Wolf, Martin, Leitenstorfer, Alfred, Huber, Rupert
Strong THz electric and magnetic fields are harnessed to coherently control charge and spin in solids with sub-cycle resolution. Exploiting coherent THz phonons we transiently induce and destroy spin density wave order in pnictides.
Extra-Adrenal Glucocorticoid Synthesis in the Intestinal Mucosa : Between Immune Homeostasis and Immune Escape
2019-06-25, Ahmed, Asma, Schmidt, Christian, Brunner, Thomas
Glucocorticoids (GCs) are steroid hormones predominantly produced in the adrenal glands in response to physiological cues and stress. Adrenal GCs mediate potent anti-inflammatory and immunosuppressive functions. Accumulating evidence in the past two decades has demonstrated other extra-adrenal organs and tissues capable of synthesizing GCs. This review discusses the role and regulation of GC synthesis in the intestinal epithelium in the regulation of normal immune homeostasis, inflammatory diseases of the intestinal mucosa, and the development of intestinal tumors.
Immunoproteasome Inhibition Impairs T and B Cell Activation by Restraining ERK Signaling and Proteostasis
2018-10-26, Schmidt, Christian, Berger, Thilo, Gröttrup, Marcus, Basler, Michael
Immunoproteasome (IP) inhibition holds potential as a novel treatment option for various immune-mediated pathologies. The IP inhibitor ONX 0914 reduced T cell cytokine secretion and Th17 polarization and showed pre-clinical efficacy in a range of autoimmune disorders, transplant-allograft rejection, virus-mediated tissue damage, and colon cancer progression. However, the molecular basis of these effects has remained largely elusive. Here, we have analyzed the effects of ONX 0914 in primary human and mouse lymphocytes. ONX 0914-treatment impaired primary T cell activation in vitro and in vivo. IP inhibition reduced ERK-phosphorylation sustainment, while leaving NF-κB and other signaling pathways unaffected. Naïve T and B cells expressed nearly exclusively immuno- or mixed proteasomes but no standard proteasomes and IP inhibition but not IP-deficiency induced mild proteostasis stress, reduced DUSP5 expression and enhanced DUSP6 protein levels due to impaired degradation. However, accumulation of DUSP6 did not cause the reduced ERK-phosphorylation in a non-redundant manner. We show that broad-spectrum proteasome inhibition and immunoproteasome inhibition have distinct effects on T cell activation at the molecular level. Notably, ONX 0914-treated T cells recovered from proteostasis stress without apoptosis induction, apparently via Nrf1-mediated up-regulation of standard proteasomes. In contrast, B cells were more susceptible to apoptosis after ONX 0914-treatment. Our data thus provide mechanistic insights how IP inhibition functionally impedes T and B cells likely accounting for its therapeutic benefits.
Few-cycle Synthetic Multi-THz Waveforms with Asymmetric Temporal Envelope
2013, Seletskiy, Denis V., Schmidt, Christian, Mayer, Bernhard, Pashkin, Alexej, Leitenstorfer, Alfred
Intense few-cycle synthetic multi-THz waveforms with strongly symmetry-broken temporal envelope are generated and field-resolved. Access to these waveforms sets an exciting platform for novel experiments in THz nonlinear optics.
Immunoproteasome Function in Lymphocyte Activation
2019, Schmidt, Christian
The immunoproteasome constitutes a specialized form of the proteasome, a multimeric protein complex with important functions in eukaryotic cells. Within a 20S core particle three different protease types cleave the polypeptide chains destined for degradation. Each of the three proteases constitutes one position per half-core particle. The standard proteasome active subunits β1c, β2c and β5c are replaced by alternative subunits in immunoproteasomes: LMP2 is incorporated at the position of β1c, MECL-1 substitutes for β2c and LMP7 substitutes for β5c. The immunoproteasome is expressed in cells of hematopoietic origin as well as in peripheral tissues under the influence of pro-inflammatory cytokines, mainly interferon-γ. Besides a well characterized role in MHC-I antigen processing, a potential role of the immunoproteasome as a drug target for treatment of autoimmune diseases was shown, as immunoproteasome inhibition proved to have disease ameliorating effects in several pre-clinical models for autoimmunity. Since the description of the immunoproteasome-selective inhibitor ONX 0914 in 2009, the underlying mechanism in the course of these autoimmune-diseases has remained largely elusive. Previous studies have shown that intrinsic effects in activated T cells, which are involved in many autoimmune disaeses, are likely to be important for the underlying mechanism. Based on previous work, which was performed during the course of my master’s thesis 2013, the aim of this study was to characterize the effects of ONX 0914 treatment at the molecular level in more detail in order to improve our understanding of the underlying mechanism of immunoproteasome inhibition. In this work, primary T and B cells from mice and humans were functionally investigated. Previous results from my master’s thesis like ameliorated CD69 up-regulation upon activation after ONX 0914 treatment were corroborated and additionally the effect was shown in B cells, in human cells and in antigenspecifically activated T cells in vivo. Furthermore, the effects were not only phenotypically characterized but substantiated by analysis at the mRNA level. An extended investigation into canonical signaling pathways of T cell activation indicated a reduction in phosphorylation of the kinase ERK after treatment with ONX 0914. This reduction was corroborated using quantitative near-infrared-dye based immunoblotting, flow cytometry and finally by confocal microscopy. However, the direct up-stream kinase was not found to be affected. Therefore, dual specificity phosphatases were identified as potential candidates for a functional involvement and several dual specificity phosphatases were analyzed at the protein level. Two of the analyzed phosphatases were affected by ONX 0914 treatment. The dual specificity phosphatase DUSP5 was less expressed, while DUSP6 accumulated at protein level. In combined cycloheximid and radioactive labelling approaches it was shown that DUSP6 degradation was impaired, but not fully blocked by ONX 0914 in T cell activation. Therfore, the possible involvement of DUSP6 for impaired T cell activation was investigated using DUSP6-deficient mice. However, it was found that DUSP6 was not responsible for the observed effects in a non-redundant manner and 11 compensation by other phosphatases cannot be ruled out. Therefore, the mechanism leading to reduced ERK-phosphorylation could not be fully unraveled so far. Apart from altered T cell signaling, the second focus of this work was set on proteostasis regulation during T cell activation after ONX 0914 treatment. Unlike previously investigated T cell lines, primary T cells and B cells showed ubiquitin-conjugate accumulation after activation when cells had been pre-treated with ONX 0914. Two factors were identified likely underlying this effect: First, it was found that almost all proteasomes in T cells and B cells constituted of LMP7-containing immunoproteasomes or mixed proteasomes. Second, it was found that the reportedly LMP7-selective inhibitor ONX 0914 co-inhibited LMP2 as well. As activated lymphocytes show marked metabolic and proteomic re-organization, these features render the cells susceptible to proteostasis stress after immunoproteasome inhibition. Consequently, the effect of enhanced proteostasis stress on activation and cell viability was characterized. It was found that T cells could alleviate the enhanced ubiquitin-conjugates within 20 hours of activation without significant induction of the integrated stress response or apoptosis. Accompanying, an enhanced neosynthesis of the standard proteasome subunit β5c and accumulation of soluble Nrf1 were detected in cell lysates. These results indicate that T cells survive mild proteostasis stress after ONX 0914 treatment likely via Nrf1-mediated proteasome up-regulation, while functional T cell activation is impaired at the same time. Similar effects were found in B cells, but induction of apoptosis-markers after ONX 0914 treatment was detected in B cells. Taken together, this work provides evidence that ONX 0914 treatment, but not immunoproteasomedeficiency, results in mild proteostasis stress in activated lymphocytes, impeding their functional capacity. This effect is likely attributed to the particularly high dependency of primary lymphocytes on LMP7- containing proteasomes and co-inhibition of LMP2 by ONX 0914. The provided mechanism hence likely explains at least parts of the anti-inflammatory effects of immunoproteasome inhibitors and why immunoproteasome inhibition shows high clinical potential with less overall toxicity as compared to broad-spectrum proteasome inhibitors.
The immunoproteasome : a novel drug target for autoimmune diseases
2015, Basler, Michael, Mundt, Sarah, Bitzer, Annegret, Schmidt, Christian, Gröttrup, Marcus
The immunoproteasome, a special class of the proteasome, is mainly expressed in cells of haematopoietic origin. Additionally, during inflammation, the immunoproteasome is induced by IFN-γ or TNF-α. In recent years it became apparent that the immunoproteasome has important functions other than processing proteins for MHC class I restricted presentation. The immunoproteasome plays a critical role in T cell expansion, cytokine production, and T helper cell differentiation. Inhibition of the immunoproteasome ameliorated disease symptoms in different animal models for autoimmune diseases. Hence, the unique role for LMP7 in controlling pathogenic immune responses provides a therapeutic rationale for targeting LMP7 in autoimmune disorders. In this review we summarise the effect of immunoproteasome inhibition in animal models for rheumatoid arthritis, inflammatory bowel disease, Hashimoto’s thyroiditis, systemic lupus erythematosus, and multiple sclerosis.
Non-perturbative Four-wave Mixing in Bulk InSb Driven by Intense Off-resonant THz Pulses
2012, Junginger, Friederike, Schmidt, Christian, Mayer, Boris, Maehrlein, S., Schubert, O., Pashkin, A., Huber, Rupert, Leitenstorfer, Alfred
A high-field multi-THz source is used to study the nonlinear response of InSb under off-resonant excitation. Field-resolved four-wave mixing signals demonstrate clear signatures of a non-perturbative regime in agreement with numerical simulations.