Guselkumab for Pityriasis Rubra Pilaris and Dysregulation of IL-23/IL-17 and NFkB Signaling: A Nonrandomized Trial.

Importance: There is no US Food and Drug Administration-approved treatment for pityriasis rubra pilaris (PRP), and it is common for patients to fail to experience improvement with several systemic options. Involvement of interleukin (IL) 23 suggests a potential therapeutic target. Objective: To determine whether guselkumab, an IL-23p19 inhibitor, provides clinical improvement for participants with PRP and better understand gene and protein dysregulation in PRP. Design, Setting, and Participants: This single-arm, investigator-initiated nonrandomized trial was conducted from October 2019 to August 2022 at a single-center academic university with participants from 8 states in the US. In total, 14 adults with moderate to severe PRP were enrolled; 12 completed the trial. Age-matched and sex-matched healthy controls provided skin and blood for proteomic and transcriptomic studies. The primary outcome was observed at 24 weeks, and additional follow-up occurred at 36 weeks. Intervention: Guselkumab is a fully human immunoglobulin G1 λ monoclonal antibody that selectively binds and inhibits the p19 subunit of IL-23. Subcutaneous injections were given at the US Food and Drug Administration-approved dosing schedule for psoriasis over a 24-week period. Main Outcomes and Measures: The primary outcome was the mean change in the Psoriasis Area Severity Index (PASI) score at week 24. Secondary outcomes included pruritus, Dermatology Life Quality Index score, clinical response at week 36, and association with transcriptomics and proteomics expression. Results: A per-protocol analysis was performed for the cohort of 4 female and 8 male patients who had a mean (SD) age of 56.5 (18.7) years. The mean improvement in PASI score, pruritus, and Dermatology Life Quality Index score was 61.8% (P < .001), 62.3% (P = .001), and 60.2% (P < .001), respectively. Nine participants (75%) achieved a 50% improvement in PASI. Among these clinical responders, at week 36, 8 of 9 achieved PASI75, and 6 of 9 achieved PASI90. No participants had pathogenic CARD14 gene variations. There was 1 serious adverse event that was not associated with the study drug. Proteomics and gene expression profiles identified dysregulation of a predominance of inflammatory pathways (such as T helper 17 and nuclear factor κ B) in participants with PRP who later responded well to treatment with guselkumab and stronger dysregulation of keratinocyte development pathways in individuals who did not respond to guselkumab. Conclusion and Relevance: The results of this nonrandomized trial suggest that guselkumab has efficacy in treating refractory moderate to severe adult PRP. Trial Registration: ClinicalTrials.gov Identifier: NCT03975153.

MITF Is Regulated by Redox Signals Controlled by the Selenoprotein Thioredoxin Reductase 1.

TR1 and other selenoproteins have paradoxical effects in melanocytes and melanomas. Increasing selenoprotein activity with supplemental selenium in a mouse model of UV-induced melanoma prevents oxidative damage to melanocytes and delays melanoma tumor formation. However, TR1 itself is positively associated with progression in human melanomas and facilitates metastasis in melanoma xenografts. Here, we report that melanocytes expressing a microRNA directed against TR1 (TR1low) grow more slowly than control cell lines and contain significantly less melanin. This phenotype is associated with lower tyrosinase (TYR) activity and reduced transcription of tyrosinase-like protein-1 (TYRP1). Melanoma cells in which the TR1 gene (TXNRD1) was disrupted using Crispr/Cas9 showed more dramatic effects including the complete loss of the melanocyte-specific isoform of MITF; other MITF isoforms were unaffected. We provide evidence that TR1 depletion results in oxidation of MITF itself. This newly discovered mechanism for redox modification of MITF has profound implications for controlling both pigmentation and tumorigenesis in cells of the melanocyte lineage.

"Near Cure" treatment of severe acute EAE in MIF-1-deficient female and male mice with a bifunctional MHCII-derived molecular construct.

Our previous studies demonstrated increased serum levels of macrophage migration inhibitory factor (MIF-1) and its homologue, MIF-2, in males during MS progression; and that genetically high-MIF-expressing male subjects with relapsing multiple sclerosis (MS) had a significantly greater risk of conversion to progressive MS than lower-MIF-expressing males and females. However, female MS subjects with severe disease expressed higher levels of CD74, the common MIF-1/MIF-2 receptor, on blood cells. In the murine model of MS, experimental autoimmune encephalomyelitis (EAE), both male and female mice lacking MIF-1 and/or MIF-2 were clinically improved during development of moderately severe disease, thus implicating both homologs as co-pathogenic contributors. The current study using MIF-deficient mice with severe acute EAE revealed a highly significant reduction of EAE scores in MIF-1-deficient females, in contrast to only minor and delayed reduction of clinical signs in MIF-1-deficient males. However, clinical EAE scores and factor expression were strongly suppressed in males and further reduced in females after treatment of WT and MIF-1-, MIF-2- and MIF-1/2-DUAL-deficient female and male mice with a MHCII DRα1-MOG-35-55 molecular construct that competitively inhibits MIF-1 & MIF-2 signaling through CD74 as well as T cell activation. These results suggest sex-dependent differences in which the absence of the MIF-1 and/or MIF-2 genotypes may permit stronger compensatory CD74-dependent EAE-inducing responses in males than in females. However, EAE severity in both sexes could still be reduced nearly to background (a "near cure") with DRα1-MOG-35-55 blockade of compensatory MIF and CD74-dependent factors known to attract peripheral inflammatory cells into the spinal cord tissue.

Brief report: Enhanced DRα1-mMOG-35-55 treatment of severe EAE in MIF-1-deficient male mice.

Macrophage migration inhibitory factor (MIF-1) and its homologue d-dopachrome tautomerase (MIF-2) share the common CD74 receptor and function innately to enhance severity of multiple sclerosis (MS) as well as the experimental autoimmune encephalomyelitis (EAE) model for MS. We previously demonstrated that genetically high-MIF-expressing male subjects with relapsing MS had a significantly greater risk of conversion to progressive MS (PMS) than lower-MIF-expressing males. To expand on this observation, we utilized MIF-1, MIF-2, and MIF-1/2-DUAL-deficient male mice to discern if there would be a greater contribution of these inflammatory factors in EAE mice with severe vs. moderate clinical disease signs. As shown previously, mice deficient in either MIF-1 or MIF-2 each had a ∼25% reduction of moderate EAE compared to WT mice, with significant differences in disease onset and trajectory. However, EAE induction in mice deficient in both MIF-1 and MIF-2 genes did not result in a further reduction in EAE severity. This result suggests that the two MIF homologues were likely affecting the same pathogenic pathways such that each could partially compensate for the other but not in an additive or synergistic manner. However, MIF-1-KO, MIF-2-KO, and MIF-1/2-DUAL-KO mice with severe EAE did not exhibit a significant reduction in cumulative EAE scores compared with WT mice, but the MIF-1-KO and, to a lesser extent, MIF-1/2-DUAL-KO mice did show a significant reduction in daily EAE scores over the last 3 days of observation, and MIF-2-KO mice showed a more modest but still consistent reduction over the same span. Furthermore, deletion of MIF-1 resulted in a massive reduction in the expression of EAE- and Complete Freund's Adjuvant-associated inflammatory factors, suggesting delayed involvement of the MIF/CD74 axis in promoting disease expression. To further explore modulation of MIF-1 and MIF-2 effects on EAE, we treated WT mice with moderate EAE using DRα1-mMOG-35-55, an inhibitor of CD74 that blocks both MIF-1 and MIF-2 action. This treatment reduced ongoing moderate EAE severity in excess of 25%, suggesting efficient blockade of the MIF/CD74 axis in disease-enhancing pathways. Moreover, DRα1-mMOG-35-55 treatment of mice with severe EAE strongly reversed EAE- and CFA-associated expression of inflammatory cytokines and chemokines including Tnf, Ccr7, Ccr6, Ccl8, Cxcr3, and Ccl19 in MIF-deficient mouse genotypes, and also exceeded innate MIF-1 and MIF-2 EAE enhancing effects, especially in MIF-1-KO mice. These results illustrate the therapeutic potential of targeting the disease-enhancing MIF/CD74 pathway in male mice with moderate and severe EAE, with implications for treatment of high-MIF-expressing RRMS human males at risk of conversion to progressive MS as well as those that have already transitioned to PMS.

Sex differences in EAE reveal common and distinct cellular and molecular components.

Experimental autoimmune encephalomyelitis (EAE) is commonly used as an animal model for evaluating clinical, histological and immunological processes potentially relevant to the human disease multiple sclerosis (MS), for which the mode of disease induction remains largely unknown. An important caveat for interpreting EAE processes in mice is the inflammatory effect of immunization with myelin peptides emulsified in Complete Freund's Adjuvant (CFA), often followed by additional injections of pertussis toxin (Ptx) in some strains to induce EAE. The current study evaluated clinical, histological, cellular (spleen), and chemokine-driven processes in spinal cords of male vs. female C57BL/6 mice that were immunized with mouse (m)MOG-35-55/CFA/Ptx to induce EAE; immunized with saline/CFA/Ptx only (CFA, no EAE); or were untreated (Naïve, no EAE). Analysis of response curves utilized a rigorous and sophisticated methodology to parse and characterize the effects of EAE and adjuvant alone vs. the Naive baseline responses. The results demonstrated stronger pro-inflammatory responses of immune cells and their associated cytokines, chemokines, and receptors in male vs. female CFA and EAE mice that appeared to be offset partially by increased percentages of male anti-inflammatory, regulatory and checkpoint T cell, B cell, and monocyte/macrophage subsets. These sex differences in peripheral immune responses may explain the reduced cellular infiltration and differing chemokine profiles in the Central Nervous System (CNS) of male vs. female CFA immunized mice and the reduced CNS infiltration and demyelination observed in male vs. female EAE groups of mice that ultimately resulted in the same clinical EAE disease severity in both sexes. Our findings suggest EAE disease severity is governed not only by the degree of CNS infiltration and demyelination, but also by the balance of pro-inflammatory vs. regulatory cell types and their secreted cytokines and chemokines.

Evaluation of Ixekizumab Treatment for Patients With Pityriasis Rubra Pilaris: A Single-Arm Trial.

Importance: Pityriasis rubra pilaris is a rare and disabling cutaneous disease that is frequently recalcitrant to conventional therapies and appears to involve interleukin (IL)-17 overexpression. Objective: To investigate the clinical response and safety of ixekizumab in treating pityriasis rubra pilaris. Design, Setting, and Participants: Single-arm, investigator-initiated trial conducted in adult patients with moderate to severe pityriasis rubra pilaris at a single-center academic university from June 2018 to January 2020. A total of 41 patients were screened, 12 were enrolled, and 11 completed the full duration of therapy. A referred, consecutive sample was used during participant selection. The treatment period and primary outcome occurred over 24 weeks with additional patient follow-up through 36 weeks. Intervention: Subcutaneous administration of ixekizumab, a humanized IgG4 antibody that binds IL-17A, at the US Food and Drug Administration-approved dosing schedule for treatment of psoriasis for 24 weeks. Main Outcomes and Measures: The primary outcome was the mean change in Psoriasis Area and Severity Index at 24 weeks. Secondary outcomes included change in affected body surface area, quality of life, induction of sustained remission, and association of improvement with CARD14 genetic variations and cutaneous cytokine expression. Results: A total of 12 white patients (mean [SD] age, 49.8 [15.1] years; 8 male [67%]) were enrolled between June 2018 and April 2019, with 11 completing the full course of intervention. The mean (SEM) improvements in Psoriasis Area and Severity Index, affected body surface area, and Dermatology Life Quality Index were 15.2 (2.1) (P < .0001), 29.8% (9.3%) (P = .009), and 9.5 (2.5) (P = .004), respectively. The 4 participants with the most improvement in Psoriasis Area and Severity Index at week 24 stayed in remission at week 36 (defined as lack of increase in Psoriasis Area and Severity Index from week 24 through week 36), off therapy. Relative dermal IL-17A expression decreased by a 1.9 log-fold change. No participants had known pathogenic CARD14 variations. There were no serious adverse events. Conclusions and Relevance: In this single-armed trial, ixekizumab was associated with reduced clinical signs and symptoms of pityriasis rubra pilaris in a subset of patients, including those in whom other systemic therapies have failed. Trial Registration: ClinicalTrials.gov Identifier: NCT03485976.

Estrogen-induced compensatory mechanisms protect IL-10-deficient mice from developing EAE.

BACKGROUND: IL-10 knockout (KO) mice are protected from experimental autoimmune encephalomyelitis (EAE) with low-dose estrogen (E2) treatment similar to wild-type (WT) mice. Previous studies have demonstrated a decrease in tumor necrosis factor in all E2-treated groups, which led to the protection of the mice. METHODS: This study used IL-10 KO mice and WT mice treated either with E2 or sham pellets 7 days prior to induction of EAE. Mice were observed for 21 days post-immunization. The spleen, inguinal lymph nodes, and brain were evaluated by flow cytometry. Spinal cords were evaluated using a cytokine/chemokine array, RT-PCR, and histology. RESULTS: This study demonstrates that E2 treatment induced three heightened regulatory mechanisms that potentially protect IL-10 KO mice from EAE: (1) an increase in programmed death-ligands 1 and 2 on monocytes and macrophages in the periphery and within the CNS; (2) an increase in CD73 in the inflamed CNS, which can increase the production of the anti-inflammatory molecule adenosine; and (3) a decrease in CD4+CD25+FoxP3+ regulatory T cells in the spleen. Together, these factors comprise an alternative compensatory mechanism that significantly downregulates key pro-inflammatory cytokine, chemokine, and chemokine receptor genes which are enhanced in the spinal cord of IL-10 KO mice. This group of E2-treated mice remained asymptomatic after EAE challenge similar to E2-treated WT mice, despite their having more T and B lymphocytes in the brain, and modestly increased demyelination in the spinal cord. CONCLUSION: These results indicate that previously unrecognized compensatory mechanisms of EAE protection are stimulated by E2 in the absence of IL-10, which can provide disease protection comparable to the IL-10-dependent effects induced by E2 in WT mice.

Increased CD74 binding and EAE treatment efficacy of a modified DRα1 molecular construct.

Multiple sclerosis (MS) is a demyelinating and degenerative disease of the central nervous system (CNS) with a strong inflammatory component that affects more than 2 million people worldwide (and at least 400,000 in the United States). In MS, macrophage migration inhibitory factor (MIF) and D-dopachrome tautomerase (D-DT) enhance the inflammatory event as a result of their interaction with their cognate receptor CD74. Therefore, the search for new agents aimed at blocking this interaction is critical for therapeutic purposes and will be of paramount importance for the treatment of MS. DRα1-MOG-35-55 constructs have been demonstrated to be effective in the treatment of experimental autoimmune encephalomyelitis (EAE) a mouse model for MS. This effect is directly correlated with the binding to its cell surface receptor, CD74, apparently preventing or blocking the binding of two inflammatory factors, MIF and D-DT. Here we report that a single amino acid substitution (L50Q) in the DRα1 domain of the human and mouse DRα1-MOG-35-55 constructs (notated as DRhQ and DRmQ, respectively) possessed increased affinity for CD74, a greater capacity to block MIF binding, the ability to inhibit pERK1/2 signaling and increased therapeutic activity in mice with EAE. These data suggest that binding affinity for CD74 could serve as an in vitro indicator of biological potency of DRhQ and thus support its possible clinical utility as an effective therapy for MS and perhaps other diseases in which there is an inflammatory reaction driven by MIF and D-DT.

Antibiotics protect against EAE by increasing regulatory and anti-inflammatory cells.

A seven day pretreatment course of an oral antibiotic cocktail (Ampicillin, Metronidazole, Neomycin Sulfate, and Vancomycin) was shown to induce changes in peripheral immune regulation and protect mice from signs of experimental autoimmune encephalomyelitis (EAE). To determine if a shorter course of antibiotic pretreatment could also protect the mice from EAE and induce regulatory immune cells, studies were conducted using the same oral antibiotic cocktail for three days. In addition, the CNS was examined to determine the effects of antibiotic pretreatment on EAE disease course and immune modulation within the affected tissue. The shorter three day pretreatment course was also significantly protective against severe EAE in C57BL/6 mice. Moreover, our study found increased frequencies of regulatory cells and a decrease in the frequency of anti-inflammatory macrophages in the spleen of EAE protected mice. Additionally, a chemokine and chemokine receptor array run on mRNA from spinal cords revealed that genes associated with regulatory T cells and macrophage recruitment were strongly upregulated in the antibiotic pretreated mice. Additional RT-PCR data showed genes associated with anti-inflammatory microglia/macrophages were upregulated and pro-inflammatory genes were downregulated. This suggests the macrophages recruited to the spinal cord by chemokines are subsequently polarized toward an anti-inflammatory phenotype. These results lend strong support to the conclusion that a three day course of antibiotic treatment given prior to the induction of severe EAE profoundly protected the mice by inducing regulatory lymphocytes in the periphery and an anti-inflammatory milieu in the affected spinal cord tissue.

MIF and D-DT are potential disease severity modifiers in male MS subjects.

Little is known about mechanisms that drive the development of progressive multiple sclerosis (MS), although inflammatory factors, such as macrophage migration inhibitory factor (MIF), its homolog D-dopachrome tautomerase (D-DT), and their common receptor CD74 may contribute to disease worsening. Our findings demonstrate elevated MIF and D-DT levels in males with progressive disease compared with relapsing-remitting males (RRMS) and female MS subjects, with increased levels of CD74 in females vs. males with high MS disease severity. Furthermore, increased MIF and D-DT levels in males with progressive disease were significantly correlated with the presence of two high-expression promoter polymorphisms located in the MIF gene, a -794CATT5-8 microsatellite repeat and a -173 G/C SNP. Conversely, mice lacking MIF or D-DT developed less-severe signs of experimental autoimmune encephalomyelitis, a murine model of MS, thus implicating both homologs as copathogenic contributors. These findings indicate that genetically controlled high MIF expression (and D-DT) promotes MS progression in males, suggesting that these two factors are sex-specific disease modifiers and raising the possibility that aggressive anti-MIF treatment of clinically isolated syndrome or RRMS males with a high-expresser genotype might slow or prevent the onset of progressive MS. Additionally, selective targeting of MIF:CD74 signaling might provide an effective, trackable therapeutic approach for MS subjects of both sexes.

Estrogen protection against EAE modulates the microbiota and mucosal-associated regulatory cells.

Sex hormones promote immunoregulatory effects on multiple sclerosis. In the current study we evaluated the composition of the gut microbiota and the mucosal-associated regulatory cells in estrogen or sham treated female mice before and after autoimmune encephalomyelitis (EAE) induction. Treatment with pregnancy levels of estrogen induces changes in the composition and diversity of gut microbiota. Additionally, estrogen prevents EAE-associated changes in the gut microbiota and might promote the enrichment of bacteria that are associated with immune regulation. Our results point to a possible cross-talk between the sex hormones and the gut microbiota, which could promote neuroprotection.

Estrogen protects both sexes against EAE by promoting common regulatory cell subtypes independent of endogenous estrogen.

Autoimmune diseases including multiple sclerosis predominantly affect females. Although high levels of sex hormones, particularly estrogen (E2), can reduce proinflammatory immune responses, it remains unclear if a lack of endogenous sex hormones might affect treatment with exogenous sex hormones. Pretreatment with E2 almost completely prevents intact female and male mice from developing clinical and histological signs of experimental autoimmune encephalomyelitis (EAE) by promoting various regulatory immune cell phenotypes. To evaluate the effects of exogenous estrogen in the absence of endogenous sex hormones, the current study compared EAE severity and the emergence of different immunoregulatory cell populations after E2 pretreatment of ovariectomized (OVX) female versus male mice. We found that E2 equally protected both OVX females and males from EAE over a 21 day observation period concomitant with reduced total cell numbers in spleen and spinal cord (males only), but enhanced percentages of CD19+CD5+CD1dhi, CD19+CD138+CD44hi and CD19+Tim-1+ Breg cells, CD8+CD122+ Treg cells and CD11b+CD 206+ARG-1+ anti-inflammatory M2-like monocytes/macrophages in both groups. In contrast, E2 decreased the percentage of CD4+CD25+FoxP3+ Treg cells in OVX females but increased these Treg cells in males and intact female mice. These data suggest that with the exception of CD4+CD25+FoxP3+ Treg cells, E2 protection against EAE promotes highly overlapping immunoregulatory subsets in OVX females and males.

Sex differences in regulatory cells in experimental stroke.

Stroke is the leading cause of disability in the United States. Sex differences, including smaller infarcts in females and greater involvement of immune-mediated inflammation in males may affect the efficacy of immune-modulating interventions. To address these differences, we sought to identify distinct stroke-modifying mechanisms in female vs. male mice. The current study demonstrated smaller infarcts and increased levels of regulatory CD19+CD5+CD1dhi B10 cells as well as anti-inflammatory CD11b+CD206+ microglia/macrophages in the ipsilateral vs. contralateral hemisphere of female but not male mice undergoing 60min middle cerebral artery occlusion followed by 96h of reperfusion. Moreover, female mice with MCAO had increased total spleen cell numbers but lower B10 levels in spleens. These results elucidate differing sex-dependent regulatory mechanisms that account for diminished stroke severity in females and underscore the need to test immune-modulating therapies for stroke in both males and females.

Sex-dependent treatment of chronic EAE with partial MHC class II constructs.

BACKGROUND: One of the main challenges in treating multiple sclerosis (MS) is reversing the effects of accumulated damage in the central nervous system (CNS) of progressive MS subjects. While most of the available drugs for MS subjects are anti-inflammatory and thus are limited to relapsing-remitting MS subjects, it is not clear to what extent their effects are capable of inducing axonal repair and remyelination in subjects with chronic MS. METHODS: A chronic model of experimental autoimmune encephalomyelitis (EAE) was used to evaluate the potency of partial MHC (pMHC) class II constructs in treating progressive EAE. RESULTS: We demonstrated an estrogen receptor alpha (ERα)-dependent increased dose requirement for effective treatment of female vs. male mice using pMHC. Such treatment using 100-µg doses of RTL342M or DRα1-mMOG-35-55 constructs significantly reversed clinical severity and showed a clear trend for inhibiting ongoing CNS damage, demyelination, and infiltration of inflammatory cells into the CNS in male mice. In contrast, WT female mice required larger 1-mg doses for effective treatment, although lower 100-µg doses were effective in ovariectomized or ERα-deficient mice with EAE. CONCLUSIONS: These findings will assist in the design of future clinical trials using pMHC for treatment of progressive MS.

Novel feedback loop between M2 macrophages/microglia and regulatory B cells in estrogen-protected EAE mice.

Immunoregulatory sex hormones, including estrogen and estriol, may prevent relapses in multiple sclerosis during pregnancy. Our previous studies have demonstrated that regulatory B cells are crucial for estrogen-mediated protection against experimental autoimmune encephalomyelitis (EAE). Herein, we demonstrate an estrogen-dependent induction of alternatively activated (M2) macrophages/microglia that results in an increased frequency of regulatory B cells in the spinal cord of estrogen treated mice with EAE. We further demonstrate that cultured M2-polarized microglia promote the induction of regulatory B cells. Our study suggests that estrogen neuroprotection induces a regulatory feedback loop between M2 macrophages/microglia and regulatory B cells.

Estrogen induces multiple regulatory B cell subtypes and promotes M2 microglia and neuroprotection during experimental autoimmune encephalomyelitis.

Sex hormones promote immunoregulatory effects on multiple sclerosis. The current study evaluated estrogen effects on regulatory B cells and resident CNS microglia during experimental autoimmune encephalomyelitis (EAE). Herein, we demonstrate an estrogen-dependent induction of multiple regulatory B cell markers indicative of IL-10 dependent as well as IFN-γ dependent pathways. Moreover, although estrogen pretreatment of EAE mice inhibited the infiltration of pro-inflammatory cells into the CNS, it enhanced the frequency of regulatory B cells and M2 microglia. Our study suggests that estrogen has a broad effect on the development of regulatory B cells during EAE, which in turn could promote neuroprotection.