Ryan Ferrie ChemicalBio 441 Week 9 Blog Spotlight
Basler, M., Mundt, S., Muchamuel, T., Moll, C., Jiang, J., Groettrup, M., and Kirk, C.J. (2014). Inhibition of the immunoproteasome ameliorates experimental autoimmune encephalomyelitis. EMBO Mol. Med. 6, 226-238. doi: 10.1002/emmm.201303543
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Multiple sclerosis (MS) is result of chronic immune mediated inflammation in the brain and spinal cord that degrades myelin sheaths. The loss of insulin disrupts the progression of action potentials through neurons and has observable phenotypic consequences. MS has been characterized by damage to important areas of white matter. Other researchers have created a model to study MS by inducing experimental autoimmune encephalomyelitis (EAE). EAE can be described as triggered brain inflammation by increased permeability of the BBB and administration of antigens into the brain, ultimately resulting in myelin deterioration. Overactive T-cells lose their affinity to bind antigens and instead induce inflammation in the brain by binding with self.
Proteins are typically destroyed following ubiquitination by large protease complexes called proteasomes. The 20S proteasome is a barrel structure with two outter rings composed of alpha subunits and two inner rings composed of beta subunits (7 subunits each ring). The catalytic site of proteolysis is found in the interior beta subunits at serine and threonine residues. Immunoproteasome is the name of the product when the catalytic subunits on regular proteasomes are replaced. Immunoproteasomes have been implicated in T cell overactivation and autoimmune diseases. In previous studies, a protein called ONX 0914 was found to reduce cytokine production from overactive T-cells, which decreased the progression of a variety of diseases in mice. ONX 0914 was able to hinder overactive T-cells by acting as an LMP7-selective epoxyketone inhibitor, which is to say that ONX 0914 lowers the activity of immunoproteasomes by blocking one of their substituted active site subunits (LMP7).
The purpose of this experiment was to take the mouse model of EAE and treat it with ONX to confirm or deny previous hypotheses about ONX. The researchers are in search of a treatment or management for multiple sclerosis in humans.
The researchers performed a number of tests on their mice to validate their findings. The authors looked into whether any of their mice were susceptible to EAE, and their controls were found not to be. In order to determine that ONX effectively acted as an LMP7 selective inhibitor, the authors immunized their mice, and then treated them with ONX three times a week. Inflammation from spinal tissue was observed microscopically. To see how ONX altered cytokine expression after EAE induction, mRNA levels for a variety of interleukines were recorded after 19 days with real time RT-PCR. This allowed them to show that ONX treated mice showed significantly lower levels of cytokines. The authors used similar methodology to show that their results are reproducible in PLP139-151 and MOG35-55 cell types.
Figure 3 (below) illustrates that inhibition of LMP7 by ONX can cause a decreased passage of T-cells through the brain, and activated lymphocyte and myeloid cells, which would contribute to the building of recently damaged areas. Furthermore, cross-sections of spinal cord were taken and stained to observe that ONX treated mice showed less severe levels of inflammation.
The best summarization of the ability of ONX to lessen the severity of EAE symptoms is Figure 6A&B (below). In this example, the authors induced EAE, but did not administer ONX until the organisms showed symptoms. Once this happened, they were treated with varying concentrations of ONX, and the figure shows that a greater dose, but not necessarily more frequent dosing, signficantly lessened the effects of the disease from that of the control.
Figure 7 (below) also gives a nice tissue stain, qualitatively showing how well ONX can reduce inflamation. Figures 6 & 7 indicate the same results in two different cell types.
The researchers found that ONX reduced symptoms of EAE and prevented brain and spinal cord inflammation. ONX is believed to be selectively targeting chymotrypsin-like activity in cells originating from the hematopoietic system. This system is the one that is mostly responsible for immunoproteasomes and the induction of EAE. Since this enzyme is so selective, there are minimal side effects associated with its treatment. The effective dose used in this study, 10 mg/kg, is below the ‘maximal tolerated’ dose of 30 mg/kg, lending to safe use of this enzyme. ONX was found not only to prevent progression of the disease, but also to inhibit its onset.
The results are significant because these authors were able to demonstrate the effectiveness of previously proposed, but never executed interactions between ONX 0914 and immunoproteasomes. While these results are significant, and were produce in vivo, experimentation was nonetheless performed on mice. Results in mice do not always translate well into the same results found in humans. The next step in this study is obviously to experiment in higher order organisms to ultimately develop an effective treatment for humans.