Immunoproteasome inhibition attenuates experimental psoriasis

Introduction Psoriasis is an autoimmune skin disease associated with multiple comorbidities. The immunoproteasome is a special form of the proteasome expressed in cells of hematopoietic origin. Methods The therapeutic use of ONX 0914, a selective inhibitor of the immunoproteasome, was investigated in Card14ΔE138+/- mice, which spontaneously develop psoriasis-like symptoms, and in the imiquimod murine model. Results In both models, treatment with ONX 0914 significantly reduced skin thickness, inflammation scores, and pathological lesions in the analyzed skin tissue. Furthermore, immunoproteasome inhibition normalized the expression of several pro-inflammatory genes in the ear and significantly reduced the inflammatory infiltrate, accompanied by a significant alteration in the αβ+ and γδ+ T cell subsets. Discussion ONX 0914 ameliorated psoriasis-like symptoms in two different murine psoriasis models, which supports the use of immunoproteasome inhibitors as a therapeutic treatment in psoriasis.


Introduction
Psoriasis is a chronic autoimmune disorder that affects 2-3% of the general population (1).It is characterized by increased keratinocyte proliferation (2), resulting in the formation of red and scaly plaques.Topical treatments, including corticosteroids, are often discontinued due to their numerous side effects (3).It is currently accepted that the disorder is mediated by the cross-talk between epidermal keratinocytes and immune cells (4).Indeed, psoriatic keratinocytes can activate neutrophils, plasmacytoid dendritic cells, and T cells (5), that aberrantly proliferate in response to inflammatory cytokines such as interleukin-22 (IL-22), and IL-17A (6).
The complexity of this disease has hampered the development of new therapies due to difficulties mimicking human psoriasis in animal models (7).Next-generation sequencing of patients with familial psoriasis revealed a gain-of-function mutation in the caspase recruitment domain family member 14 (CARD14) (8).Heterozygous mice harboring a CARD14 gain-of-function mutation (Card14DE138 +/-) spontaneously develop a chronic psoriatic phenotype with scaling skin lesions (9).Several other murine models induce psoriasis-like features (10).Topical application of imiquimod (IMQ), a TLR7/8 activator, induces skin inflammation mediated via the IL-23/IL-17A axis (11).Psoriatic lesions depict an upregulation in retinoic acid-related orphan receptor C (RORC) mRNA (12), which controls the lineage commitment of T helper type 17 (Th17) (13).The increase of IL-17A and IL-22 in serum samples of psoriasis patients (14) demonstrates that its pathogenesis is driven by the IL-23/IL-17A axis.Furthermore, the neutralization of cytokines that maintain Th17 cell polarization reduces skin lesions (9).
The immunoproteasome is a special form of the 26S proteasome in which the standard catalytically active b-subunits (b1c, b2c, and b5c) are replaced by low molecular mass polypeptide (LMP)2 (b1i), multicatalytic endopeptidase complex-like (MECL)-1 (b2i) and LMP7 (b5i).The expression of both standard proteasome and immunoproteasome subunits is increased in lesional psoriasis skin (15).The immunoproteasome is not only involved in the generation of antigenic peptides that are presented to cytotoxic T cells (16) but has a strong influence on T helper cell commitment (17).Immunoproteasome inhibition is a promising strategy in reducing IL-23 secretion and suppressing Th17 cell development (18).Irreversible inhibition of the LMP2/LMP7 subunits of the immunoproteasome via treatment with ONX 0914 has been demonstrated to ameliorate several inflammatory diseases (19)(20)(21)(22).
In this study, the therapeutic potential of immunoproteasome inhibition in psoriasis pathogenesis was assessed in Card14mediated and IMQ-induced psoriasiform models.We found disease amelioration in two different pre-clinical psoriasis models, which suggests selective inhibition of the immunoproteasome as a potential therapeutic treatment strategy for psoriasis.

Murine models and proteasome inhibition
ONX 0914 (Kezar Life Sciences) was formulated in 10% sulfobutylether-b-cyclodextrin and 10 mM sodium citrate (pH 6; vehicle) (19).The administration was performed s.c. at 10 mg/ kg, which has extensively been used in the past not causing cytotoxic effects even at a higher concentration (12 mg/kg) (21).The activity of the proteasome after the use of ONX 0914 was previously investigated (19, 24).In the IMQ-induced psoriasislike model, IL-17A-GFP mice were shaved on the back and 5% IMQ cream (Aldara, MEDA) was applied to the back and the ear daily for 8 consecutive days.Starting on day 3, mice were treated daily with ONX 0914 or vehicle s.c.Experiments with Card14DE138 +/-mice started at the age of 8-10 weeks.Mice were treated with ONX 0914 or vehicle s.c. on alternate days for 20 days.

Ear thickness and skin inflammation score
Ear thickness was measured (thickness gauge; Mitutoyo) daily or on alternate days in the IL-17A-GFP and Card14DE138 +/-mice, respectively.Eczema and scaling on the ear and back were evaluated visually in a blinded manner and quantified on a range from 0 to 4 points (0, no change; 1 mild change, 2 marked change, 3 significant change, 4 severe change).The inflammation score represents the sum of both factors.

Real-time RT-PCR
RNA was extracted from the ear tissue using Trizol (ThermoFisher) according to manufacturer´s protocols.The cDNA was prepared using the Biozym cDNA conversion kit (Biozym).Afterwards, real-time RT-PCR (Biozym Blue S´Green Kit) was performed in a Biometra TProfessional Thermocycler

Histology
Hematoxylin-eosin sections were prepared as in (22).For immunofluorescence staining, the samples were flash-frozen in liquid nitrogen and embedded into anOptimal cutting temperature compound (OCT) medium.Sections of 14 mm were prepared using the Frigocut 2800E (Reichert Jung/Leica) and were hydrated in PBS at RT for 10 min.The samples were fixed with acetone at 4°C for 15 min and washed in PBS.Staining (listed in Supplementary Table 2) was performed overnight at 4°C . Counterstaining was performed with DAPI mounting medium (ThermoFisher).Images were taken in AxioImager (Zeiss).Quantification of the epidermal thickness was performed in ImageJ (U.S. National Institutes of Health) as described in (25).Quantification of the immune populations infiltrating the ear was performed by measuring the percentage of the positive area and normalizing it to DAPI with ImageJ.

Organ preparation and flow cytometry
Spleens were collected and a single cell suspension was prepared using 70 µm nylon mesh.Ears were harvested and dorsal and ventral sections were split with forceps.Digestion was performed with 1 mg/ml DNAse I (Sigma) and 1 mg/ml collagenase D (Roche) in HBSS (10 mM Hepes) in a gentleMACS Octo Dissociator (Miltenyi Biotec).Cytokine production was analyzed after restimulation with 25 ng/ml phorbol-12-myristat-13-acetat (PMA), 500 ng/ml ionomycin and 10 µg/ml brefeldin A (BFA) (all Merck) for 4 hours at 37°C , 5% CO2.Surface and intracellular staining was performed as in (26).Doublet exclusion was performed by gating on SSC-W/ SSC-H or SSC-H/SSC-A.The surface staining was performed first along with fixable viability stain 780 (BD Pharmingen) according to the manufacturer's instructions.The antibodies used are listed in Supplementary Table 2.The samples were measured on LSRFortessa (BD Biosciences).Cell count in the ear was performed using Cytoflex (Beckman coulter).Flow cytometry data was analyzed with FlowJo v10 (BD Biosciences).

Serum collection and enzyme-linked immunosorbent assay (ELISA)
Blood was collected by cardiac puncture.The analysis of IL-17A, IL-6 and TNF (ThermoFisher Scientific) was performed as in (22).

Statistics
Data is expressed as mean ± S.D and was analyzed using Prism 9.1 (Graphpad).The Shapiro-Wilk (W) test was used to verify normal distribution.Data without a normal distribution were analyzed with non-parametric tests (Kruskal-Wallis or Mann-Whitney test), and data with a normal distribution were analyzed with parametric tests (unpaired t-test, Ordinary one-way or twoway ANOVA), including the post hoc test Bonferroni, Tukey, S ̌idaḱ or Fisher´s LSD.Statistical significance was achieved when p < 0.05; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Immunoproteasome inhibition attenuated psoriasis-like lesions in
Card14DE138 +/-mice Card14DE138 +/-mice develop spontaneous ear skin lesions at approximately 8 weeks of age that mimic human psoriasis.To investigate the potential therapeutic use of ONX 0914 we treated Card14DE138 +/-mice at the age of 8-10 weeks with 10 mg/kg ONX 0914 on alternate days for 20 days (Figure 1A).Mice treated with the immunoproteasome inhibitor depicted significantly decreased ear thickness and epidermal thickness compared to vehicle-treated mice (Figures 1B, D).Furthermore, the inflammation score was significantly decreased after treatment with ONX 0914 (Figure 1C).The hematoxylin-eosin sections of the ear demonstrated the presence of thickening epidermis (acanthosis) and thickened stratum corneum (hyperkeratosis) in Card14DE138 +/-mice (Figure 1D).In contrast, treatment with ONX 0914 notably alleviated the histopathology features typical of psoriasis.We also observed an increase in the size of the draining lymph nodes (dLNs) collected from Card14DE138 +/-vehicle-treated mice in comparison to naïve mice (Figure 1E).Even though the organ weight ratio of the dLNs after immunoproteasome inhibition was not reduced to basal levels of naïve mice, a significant reduction compared to vehicle-treated mice was detected.In contrast, ONX 0914-treated mice depicted a significantly increased weight of the spleen.The percentage of IL-17A + cells were significantly reduced after treatment with ONX 0914 in both auricular and inguinal lymph nodes (Supplementary Figure 1) while the percentage of IL-22 + cells was not affected.We also investigated the presence of IL-17A-secreting CD4 + cells in the spleen (Figure 1F), which was significantly reduced in ONX-0914-treated mice.In contrast to IL-17A, the serum levels of TNF and IL-6 in Card14DE138 +/-mice were elevated compared to naïve control mice.However, no difference in serum levels of TNF and IL-6 was observed between ONX 0914-treated and vehicle-treated mice (Supplementary Figure 2).

Expression patterns of psoriasisrelated genes
To assess the changes in the inflammatory milieu, we determined the gene expression of several inflammatory mediators in the ear tissue of Card14DE138 +/-mice (Figure 2).
Compared to naïve wild-type mice, several inflammation-related ge nes were upregulat ed in Card14 D E138 + / -mic e. Immunoproteasome inhibition significantly decreased the mRNA expression of the inflammatory mediators Il17c, Tnf, Ccl20, Il22, and Il23.No differences in the expression of Il17a, Il6, or Cxcl2 were detected.

ONX 0914 reduces the inflammatory infiltration in the ear of psoriatic mice
Phenotyping the psoriasis inflammatory infiltrate revealed abundant mononuclear cells in the ear of Card14DE138 +/-mice (Figure 3).We detected the presence of CD45 + and CD4 + cells distributed along the epidermis and dermis (Figure 3A).IL-17A seemed to be confined close to the epidermis.Quantification of the immunofluorescence signal in ear sections of Card14DE138 +/-mice (Figure 3B) revealed that ONX 0914 treatment reduced the presence of CD3 + cells, CD4 + cells, and the pro-inflammatory cytokine IL-17A.
To confirm these results we investigated the inflammatory infiltrates in the ear by flow cytometry (Figure 3C).We observed a significant reduction in the absolute cell count of CD45 + , CD3 + , CD4 + , CD11b + Ly6G + , and CD4 + IL-17A + in the ear of Card14DE138 +/-mice treated with ONX 0914 and the reduction of CD8 + , CD4 + and CD19 + in the spleen (Figure 3D).The reason for the apparent discrepancy between the observed increased spleen weight (Figure 1E) and the reduction of the numbers of CD8 + , CD4 + and CD19 + in the spleen (Figure 3D) of ONX 0914 treated mice is currently unknown.

Immunoproteasome inhibition modulates the ab + and gd + T cell subsets
Skin homeostasis is maintained by balancing keratinocyte proliferation and destruction (27).In the past, most of the T cell functions have been attributed to ab + T cells, while gd + T cells have been overlooked (28).Therefore, we analyzed the presence of ab + and gd + T cells subsets in the ear.We observed that inhibition of the immunoproteasome in Card14DE138 +/-mice induced a change in the T cell pool by decreasing the percentage of ab + T cells and increasing the gd + T cells (Figures 4A,B).
Both dermal ab + and gd + T cells can secrete IL-17A and IL-23 (29), which has been linked to the pathogenesis of psoriasis (30).To dissect the cellular source of IL-17A we analyzed the secretion of IL-17A and IL-22 cytokines by ab + and gd + T cells in the ear tissue of Card14DE138 +/-mice after a short restimulation in vitro.While approximately 40% of the IL-17Asecreting cells in the ear were ab + T cells, ONX 0914 significantly decreased the secretion of IL-17A by these cells.IL-22 secretion was reduced as well in ONX 0914 treated mice (Figure 4B).This shift in IL-17A and IL-22 secretion suggests that immunoproteasome inhibition shapes the immunological response, causing an alteration in the cell subsets.Immunoproteasome inhibition reduces the expression of inflammatory genes in Card14DE138 +/-mice: 8-10 weeks old Card14DE138 +/-mice were treated on alternate days with 10 mg/kg ONX 0914 (n = 7) or vehicle (n = 6) for 20 days.Real-time RT-PCR analysis of ear tissue was performed for Il17a, Il17c, Tnf, Ccl20, Il6, Il22, Il23, and Cxcl2.On the g-axis, the relative expression of each gene is depicted.Naïve C57BL/6 mice were used as a control indicated by the dotted line.Data were analyzed following the 2 -DDCt method and normalized to hprt.Data were pooled from 2 independent experiments and statistically analyzed by unpaired t-test.Values represent mean ± SD. * p < 0.05, and ** p <0.01.

ONX 0914 ameliorates the skin lesions in the psoriasis-like mouse model
To validate our findings in another murine model of psoriasis, we analyzed the effect of immunoproteasome inhibition in the IMQ-induced psoriasis-like mouse model, which is an acute psoriasiform model.To ensure proper immunoproteasome inhibition in the acute psoriasis-like mouse model ONX 0914 was administered daily instead of every second day as applied in the Card14DE138 +/-mice.To easily track IL-17A-secreting cells, IL-17A-GFP reporter mice were used.IL-17A-GFP mice received daily IMQ or vaseline cream applied on the back and the ear for 8 consecutive days (Figure 5A).ONX 0914 or vehicle was administered starting on day 3, a time point when the ear skin had significantly thickened Immune cell populations in the ear and spleen of Card14DE138 +/-mice: 8-10 week old Card14DE138 +/-mice were treated on alternate days with 10 mg/kg ONX 0914 (n=6-7) or vehicle (n=4-7).20 days after treatment, 14 mm ear cryosections were stained with anti-CD3, anti-CD4, anti-IL-17A, anti-CD11c, anti-CD45, anti-Ki67, antibodies (all in green) and DAPI (in blue).Representative images are shown.The scale bar is 50 mm (A).The positive signals were quantified with ImageJ (B) On the g-axis, the ratio of the fluorescence signal to DAPI is depicted.Data (n=4-7) were pooled from 2 independent experiments and statistically analyzed by unpaired t-test or Mann-Whitney test.(C) A single cell suspension of the ear (n=6) was prepared after 20 days of ONX 0914 or vehicle treatment and the CD45 + , CD3 + , CD11b + Ly6G + , CD4 + , and CD4 + IL-17A + populations were analyzed.On the g-axis, the absolute cell count per ear is depicted.(D) The spleen was analyzed for CD45 + , CD8 + , CD4 + , and CD19 + .The absolute cell number is depicted on the g-axis.Data were pooled from 2 independent experiments (n=6) and statistically analyzed by unpaired t-test or Mann-Whitney test.(C, D) The cells were gated on CD45 + cells after doublet and dead cell exclusion.The gating strategy for C and D is depicted in Supplementary Figure 3B Representative flow cytometry plots for C and D are depicted in Supplementary Figure 4A.All values represent mean ± SD. * p < 0.05, and ** p <0.01.
i n c o m p a r i s o n w t h d a y 0 ( F i g u r e 5 B ) .T h u s , immunoproteasome inhibition started when disease symptoms were already present, which mimics a therapeutic setup.Daily treatment with ONX 0914 or vehicle was continued until day 7 post first IMQ application (Figure 5A).The analysis of IL-17A levels in the serum revealed a significant increase of IL-17A in the IMQ-treated mice compared to vaseline-treated mice, while ONX 0914 treatment significantly reduced the IL-17A levels in the serum to values similar to vaseline-treated control mice (Figure 5C).As depicted in Figure 5A we could visually observe a reduction of the IMQ-induced lesions after immunoproteasome inhibition.Indeed, both thicknesses of the ear and the back were significantly reduced starting on day 6 (Figure 5D).The inflammation scores were reduced in both the ear and the back.However, the reduction of the skin lesions seemed to be more prominent in the ear.Furthermore, the hematoxylin-eosin sections of the ear and back (Figure 5E) demonstrated a visual reduction of the tissue thickness and local parakeratosis.An evident reduction of rete ridges, which are considered a main hallmark of psoriasis, can be observed on the back of ONX 0914treated mice.
We also observed that dLNs in IMQ-treated mice were heavier (Figure 6A).Even though no significant difference was observed for the auricular LNs, we could observe a normalization of the weight in the inguinal LNs of the mice treated with ONX 0914.Although the weight of the spleen was increased after IMQ application, ONX 0914 treatment had no influence.
The recruitment of IL-17A cells to the inflamed areas was analyzed by tracking the expression of GFP on the IVIS Spectrum in vivo imaging system.We could detect a significant increase in the GFP signal in the ear and back of IMQ-treated mice (Supplementary Figure 5), which suggests that this method can be used to track in vivo recruitment of IL-17A + cells to the skin.We detected a lower intensity of the GFP signal on day 8 post first IMQ-treatment in the ear of ONX 0914-treated mice.On the back, no difference between vehicleand ONX 0914-treated mice could be observed.Additionally, we analyzed the inflammatory infiltrate in the ear by fluorescence microscopy in the IMQ-induced psoriasis model and quantified it (Figures 6B, C).Several immune populations were detected in the ear tissue, of which CD45 + and CD3 + cells were significantly Immunoproteasome inhibition alters the ab + and gd + T cell subsets.8-10 weeks old Card14DE138 +/-mice were treated on alternate days with ONX 0914 (n = 6) or vehicle (n = 6) for 20 days.A single cell suspension of the ear was prepared and stimulated with PMA, ionomycin and BFA for 4 hours at 37°C.Then, an intracellular cytokine staining for IL-17A and IL-22 was performed.The ab + or gd + were gated on CD45 + CD3 + cells.(A) Quantification of the frequencies of the analyzed cells.On the g-axis, the percentage of the indicated population of viable or of CD45 + CD3 + cells is depicted.Representative flow cytometry plots are shown on the left panel.ab + and gd + cells were gated on CD45 + CD3 + cells after doublet and dead cell exclusion.(B) IL-17A and IL-22 secreting ab + or gd + T cells.Representative flow cytometry plots are shown on the left panel.ab + and gd + cells were gated on CD45 + and IL-17A + or IL-22 + cells after doublet and dead cell exclusion.Data were pooled from two independent experiments and analyzed by unpaired t-test (A) or two-way ANOVA followed by a S ̌idaḱ test (B).The gating strategy is depicted in Supplementary Figure 3D.Gating was performed using FMO spleen samples.IL-17A and IL-22 secretion was gated using Frontiers in Immunology frontiersin.orgreduced after treatment with ONX 0914.For CD4 + and Ki67 + a tendency to numbers could be observed.Taken together, similar to flow cytometry experiments (Figure 3) lower inflammatory infiltrates could be detected by fluorescence microscopy in ONX 0914-treated mice.

Discussion
During the last decades, intensive research on psoriasis pathogenesis has been translated into the development of potential therapies (31).However, the inconsistency in patient responses (32) and the high rate of psoriasis that remains untreated (33) highlight the need for new and treatments.In this study, we demonstrate the effective use of the immunoproteasome inhibitor ONX 0914 in reducing tissue thickness, inflammatory infiltrate, and skin damage in both Card14-mediated and IMQ-induced psoriasis.
Even though the pathogenesis of psoriasis is not fully understood, it is accepted that reactive-oxygen species (ROS) and oxidative stress contribute to disease progression (34).The resulting protein carbonylation, which was detected in patients with psoriasis (35), is irreversible and requires defective proteins to be degraded in order not to disrupt cellular metabolism (36).Such proteins are degraded mainly by the proteasome (37), which is dysregulated in many diseases (38).The analysis of skin lesions revealed that the expression of the 26S proteasome were increased and mainly detected in inflammatory clusters infiltrating the dermis (15).These results strongly support the rationale of treating psoriasis with proteasome inhibitors.
Immunoproteasome inhibitors have been widely used to treat inflammatory diseases in pre-clinical animal models (39).Therapy with broad spectrum proteasome inhibitors were effective in the treatment of psoriasis in the murine SCID-hu model ( 40) by reducing T cell activation.Although the proteasome inhibitor bortezomib was efficacious in the thioglycolate-induced MCP-1 production model, it exacerbated symptoms in the IMQ-induced psoriasis model (41).In humans, broad spectrum proteasome inhibitors have rather severe side effects, such as anemia, thrombocytopenia, and neutropenia, limiting its therapeutic Immunoproteasome inhibition normalizes the weight of dLNs and ameliorates the inflammatory infiltrate in IMQ-induced psoriasis-like inflammation.IL-17A-GFP mice were treated as described in Figure 5A.(A) The dLNs and spleens were harvested after 8 days of treatment with IMQ/vaseline.On the g-axis, the organ weight normalized to the body weight is depicted.Data (vaseline vehicle n = 4-5, IMQ vehicle, and ONX 0914 n = 6) was pooled from two independent experiments and analyzed by a one-way ANOVA followed by a S ̌idaḱ test.(B) Representative images of ear cryosections that were stained with anti-CD3, anti-CD4, anti-CD45, anti-Ki67 antibodies or IL-17A (all in green), and DAPI (in blue).The scale bar is 100 mm (C) The positive signal was quantified with ImageJ.On the g-axis, the ratio of the fluorescence signal to DAPI is depicted.Data (n = 4-6) were pooled from 2 independent experiments and statistically analyzed by unpaired t-test or Mann-Whitney test.All values represent mean ± SD. * p < 0.05, ** p <0.01, and *** p < 0.001.applicability for psoriatic diseases.However, due to the expression of immunoproteasomes in hematopoietic cells the immunoproteasome inhibitors fewer toxic side effects (42).Interestingly, the immunoproteasome inhibitor PKS3053 prevented the induction of several IFN-regulated genes and the pro-inflammatory cytokines TNF and IL-1b to tape stripping (43) in a mouse model for atopic dermatitis (44).
Psoriasis is a complex disease that cannot be fully mimicked in animal models.For this reason, we employed two distinct animal models (one chronic and one acute) for testing the efficacy of the immunoproteasome inhibitor ONX 0914.In both, the Card14and the IMQ-model, we observed the amelioration of physical manifestations of psoriasis in ONX 0914-treated mice.Interestingly, skin cell replacement takes place every 28-30 days in healthy human individuals.However, the turnover is increased to 4-7 days in psoriatic patients (45).Therefore, we analyzed cell proliferation by detecting Ki-67 + cells in the dermis and epidermis of Card14DE138 +/-mice.Even though we did not detect a significant alteration of Ki-67 + cells after treatment with ONX 0914 (Figure 3), we found cell counts of CD45 + , CD3 + , CD4 + , CD4 + IL-17A + and CD11b + Ly6G+ to be markedly reduced after treatment.
Cytokine members of the IL-23/IL-17 family are critical in the development of autoimmunity and psoriasis (46).IL-23 activates Th17 cells through the STAT3 pathway and promotes the production of IL-17A, IL-22 and TNF, which induce the proliferation of keratinocytes expressing the IL-22 receptor (47).We observed that CD4 + IL-17A + cells were significantly increased in the spleen of Card14DE138 +/-mice and subsequently diminished after immunoproteasome inhibitor treatment (Figure 1).However, such upregulation of the IL-17A cells in the spleen could not be detected in IMQtreated mice (data not shown).Cutaneous inflammation is not a problem solely related to skin, but the release of several inflammatory products into systemic circulation can affect other organs resulting in comorbidities (48).Interestingly, IL-17A is responsible for the formation of amyloidosis in both the liver and the spleen (49), a disorder in which abnormal proteins accumulate.Additionally, IL-17-related cytokines play an important function in the formation of microabscesses by neutrophils through "connection to IkB kinase and stressactivated protein kinases" signaling into the keratinocytes (50).In line with this, and contributing to the reduced disease symptoms in our study, we observed a reduction of neutrophils (CD11b + Ly6G + ) accompanied by a normalization in the cell counts of several other immune cell populations in the skin of Card14DE138 +/-mice treated with ONX 0914 (Figure 3).
gd + T cells are a particular population of T lymphocytes.Even though most of the studies have focused on ab + T cells, there is increasing evidence that aberrantly activated gd + T cells play an important role in the pathogenesis of autoimmune disorders, such as psoriasis (51).IL-23 predominantly stimulates dermal gd + T cells to produce IL-17 that leads to disease progression (29).Since both ab + and gd + T cell population have the ability to secrete IL-17A and IL-22 (52) we investigated these populations in the skin samples of diseased Card14DE138 +/-mice (Figure 4).We observed that ab + T cells are the main producers of IL-17A in the skin of Card14DE138 +/-mice, which is in line with prior analysis (53).Little IL-22 was secreted by gd + T cells in Card14DE138 +/-mice.Interestingly, ONX 0914 treatment reduced the percentage of ab + cells and ab + cells secreting IL-17A, whereas it increased the frequency of gd + T cells and IL-22 production.IL-22 is primarily involved in preservation of the mucosal barrier and protection of the host from microbial parasites in the skin (54).The anti-apoptotic effects of IL-22 (55) together with the capability to promote regeneration and proliferation highlights IL-22´s ability to promote healing and skin repair (56).Whether gd + T cells may have a protective function by increased production of IL-22 is currently unknown.Remarkably, we observed a double positive gd + ab + T cell population in the ear tissue (Figure 4B).Several non-common ab/gd TCRs have been previously reported (57)(58)(59)(60) and suggested to be produced as unusual gene rearrangements.Recently, Reitermaier et al. discovered that abgd double positive T cells are present in fetal human samples and are essential in the skin development and immunity (61).Whether these ab + gd + cells play a relevant role in our disease model is currently unknown.
Taken together, this study shows that ONX 0914 significantly reduced the skin thickness and pathological features in two different murine model of psoriasis.The analysis of skin samples revealed normalization of pro-inflammatory cytokines and cell populations that contribute to the pathogenesis of psoriasis.Moreover, the reduction of ab + T cells was accompanied by a significant shift in the IL-17A and IL-22 secretion.Altogether, this study highlights the potential therapeutic use of immunoproteasome inhibitors in the treatment of psoriasis.

1
FIGURE 1Immunoproteasome inhibition attenuated the psoriasis-like lesions in Card14DE138 +/-mice: 8-10 weeks old Card14DE138 +/-mice were treated on alternate days with 10 mg/kg ONX 0914 or vehicle for 20 days.(A) Experimental setup.(B) Ear thickness was measured with a thickness gauge.On the g-axis, the ear thickness in mm is depicted.Data (vehicle n = 7, ONX 0914 n = 6) was pooled from two independent experiments and analyzed by a two-way ANOVA followed by a S ̌idaḱ test.(C) The inflammation score was measured visually on alternate days and results from the sum of the eczema and scaling scores, which are shown on the g-axis.Data (vehicle n = 16, ONX 0914 n = 15) was pooled from five independent experiments and analyzed by a two-way ANOVA followed by a S ̌idaḱ test.(D) Representative hematoxylin-eosin-stained sections from the ear of Card14DE138 +/-after 20 days of treatment with ONX 0914 or vehicle.The epidermal thickness was measured in ImageJ and normalized to the epidermal area, which is depicted on the g-axis.Data (vehicle n = 9, ONX 0914 n = 11) was pooled from three independent experiments and analyzed by a Mann-Whitney test.The scale bar is 200 mm.(E) The auricular lymph nodes and the spleens were harvested after 20 days of ONX 0914 treatment and weighed.On the g-axis, the organ-to-body weight ratio is depicted.Naïve mice were used as controls.Data (naïve n = 11, vehicle n = 12, ONX 0914 n = 11) was pooled from three independent experiments and analyzed by one-way ANOVA followed by a Tukey´s test.(F).The splenocytes of mice treated with ONX 0914 or vehicle were collected and stimulated with PMA, ionomycin and BFA for 4 hours at 37°C.Then, an intracellular cytokine staining for IL-17A was performed.On the g-axis the frequency of IL-17A + cells in the spleen is depicted (left panel).The gating strategy is depicted in Supplementary Figure3Aand includes doublet and dead cell exclusion.The IL-17A + cells are pregated on CD45 + CD4 + cells.Gating was performed using a Fluorescence minus one (FMO) control.Representative dot plots are depicted on the right panels.Data (n = 6) was pooled from two separate experiments and analyzed by one-way ANOVA followed by a Tukey´s test.All values represent mean ± SD. * p < 0.05, ** p <0.01, *** p < 0.001, and **** p < 0.0001.

5
FIGURE 5 Immunoproteasome inhibition ameliorates IMQ-induced psoriasis-like inflammation in mice.IL-17A-GFP mice were treated with IMQ or vehicle (vaseline) on the back and the ear for 8 consecutive days.(A) Experimental setup and representative images of the back of the mice after 8 days of IMQ application.(B) Ear thickness in mm on day 0 and day 3 of IMQ-treated mice (n = 12).Data were pooled from three independent experiments and analyzed by paired t-test.(C) IL-17A levels in the serum of mice after 8 days of treatment.Data (vaseline vehicle-treated mice n = 5, IMQ vehicle and IMQ ONX 0914 n = 6) was pooled from two independent experiments and analyzed by one-way ANOVA followed by a Tukey´s test (D) Ear and back thickness were measured with a thickness gauge.On the g-axis, the ear thickness in mm is depicted.The thickness from the vaseline vehicle group is depicted for clarification (dotted line) and was not statistically analyzed.The inflammation score was measured visually on alternate days and results from the sum of the eczema and scaling scores, which is shown on the g-axis.Data (IMQ vehicle n = 6, IMQ ONX 0914 n = 6) was pooled from two independent experiments and analyzed by a two-way ANOVA followed by a S ̌idaḱ test.(E) Representative images of hematoxylin-eosin stained sections from the ear and back of vaseline-vehicle, IMQ-vehicle, or IMQ-ONX 0914-treated mice.The scale bar is 200 mm.Epidermal thickness from the ear and the back was calculated using ImageJ.Data (vaseline vehicle n = 2, IMQ vehicle, and ONX 0914 n = 5) was pooled from two independent experiments and analyzed by one-way ANOVA followed by a Tukey´s test.All values represent mean ± SD. * p < 0.05, ** p <0.01, *** p < 0.001, and **** p < 0.0001.