Pathogenic Cytokines Research Articles

Concurrent occurrence of ulcerative duodenitis and ulcerative colitis displaying unique responses to golimumab and ustekinumab.

Recent studies have reported the occurrence of upper gastrointestinal (UGI) inflammation in patients with ulcerative colitis (UC). However, whether UC-associated UGI and colorectal lesions share pathogenic cytokine profiles and responses to biologics remains unknown. Herein, we report a case of concurrent UC and ulcerative duodenitis (UD) that displayed unique responses to biologic treatment. Although treatment with prednisolone (PSL) failed to induce remission in both disorders, golimumab (GLM) and ustekinumab (UST) were effective against UD and UC, respectively, and remission of both disorders was achieved using UST. Immunofluorescence analyses revealed that numbers of immune cells expressing TNF-α were comparable in both duodenal and rectal mucosa before the treatment. GLM or UST treatment markedly decreased numbers of TNF-α-expressing duodenal immune cells, suggesting the presence of correlation between TNF-α expression and disease activity of UD. In contrast, TNF-α expression was not parallel to disease activity of UC because GLM or PSL failed to induce remission despite a marked reduction in TNF-α expression. Responsiveness to GLM or UST together with immunofluorescence studies suggests that TNF-α and IL-12/23p40 are pathogenic cytokines causing UD and UC, respectively, in the present case.

Comparative Evaluation of Soft Tissue Reaction to Titanium and Polyetheretherketone Customized Healing Abutments: a Split-Mouth Randomised Clinical Trial

Aim. This study aimed to evaluate the peri-implant soft tissue response to customized healing abutments (CHAs) made of titanium and polyetheretherketone (PEEK). Methods. Titanium and PEEK CHAs were mounted on bone-level dental implants in bilateral edentulous patients in a randomized split-mouth study design. After a three-week healing period, the clinical parameters of the peri-implant soft tissues were assessed. The value of marginal bone resorption was measured by cone-beam computed tomography. In addition, peri-implant crevicular fluid samples were obtained to identify periodontal pathogenic bacteria, cytokine levels, and active neutrophil collagenase (aMMP-8) concentration. Microcirculation and oxygenation parameters were assessed using laser Doppler flowmetry and optical tissue oximetry. In addition, wavelet analysis was conducted to study blood flow control mechanisms. Results. Out of the 23 participants initially recruited, 20 were assessed after titanium and PEEK CHA installation due to losses to follow-up. The peri-implant soft tissue around 27 titanium and 25 PEEK CHAs was evaluated during follow-ups. No differences were found in the values of marginal bone resorption, cytokine levels, or total bacterial load (p>0.05). PEEK CHAs demonstrated lower bleeding on probing values, less plaque accumulation, and faster relief of ischemia and hypoxia. Although aMMP-8 concentration was higher in the PEEK group, the values were within normal limits. Conclusions. The results of this comparative study showed a positive soft tissue response to PEEK CHAs. However, further studies comparing the effectiveness of different materials for CHAs should be carried out, considering their specific physical properties and processing protocols.

Prognostic factors and treatment outcomes in pediatric autoimmune encephalitis: a multicenter study.

The last few decades have increased our understanding of autoimmune encephalitis (AE). In both the pediatric and adult populations, it proves to be a disease of dramatic acute onset of heterogeneous clinical manifestations, notably encephalopathy with neuropsychiatric symptoms, seizures, and extrapyramidal symptoms. More often, it is triggered by a viral infection in the pediatric age groups, as suggested by the preceding febrile symptoms in over half of cases, and more ostensibly, NMDAR encephalitis post herpes encephalitis. An underlying neoplasm may be present in certain types (i.e., NMDAR encephalitis). The rising rate of antibody detection and subsequent confirmation has been boosted by improved live cellular assay detection methods. The corresponding clinical phenotypes, common underlying malignancies, and histopathological findings have helped improve our management regarding intervention and choice of immunotherapy. New assessment tools such as the Clinical Assessment Scale in Autoimmune Encephalitis (CASE score) have helped improve the objective assessment of impact on cognitive functions (1). Early intervention with immunotherapy (and tumor removal in proven underlying neoplasms) has improved overall outcomes in most presenting patients. But nearly 40% of cases fail to respond to the first tier of treatment (2). The complex interplay between pathogenic autoantibodies, T-cells, B-cells, and cytokines has led to the emergence of additional immunotherapy agents (i.e., tocilizumab and bortezomib). In this retrospective observational study of pediatric AE conducted at two tertiary care centers, we observed the clinical characteristics, autoantibody yield, treatment modalities used, and disability scores during presentation and follow-up. Our secondary aim was to delineate prognostic factors for poor outcomes. Neuropsychiatric symptoms, encephalopathy, and seizures were the predominant manifestations in most of our patients. Younger age groups, refractory seizures, profound encephalopathy, and refractory disease harbored higher disability scores. The group that received combined immunotherapy has shown mitigation of disability score from severe to mild during long-term follow-up, signifying the role of multifaceted immunotherapy in pediatric refractory AE. Early implementation of combined immunotherapy in refractory cases significantly improved longterm disability scores, in spite of lingering residual effects on neurologic functions, notably cognition, behavior, and speech.

The role of lipid peroxidation in epithelial–mesenchymal transition of retinal pigment epithelial cells

Epithelial–Mesenchymal Transition (EMT) of retinal pigment epithelial (RPE) cells is recognized as pivotal in various retinal diseases. Previous studies have suggested a reciprocal regulation between reactive oxygen species (ROS) and EMT, though the involvement of peroxidized lipids or the effects of reducing them has remained unclear. The present study disclosed that EMT of ARPE-19 cells induced by TGF-β2 and TNF-α involves increased lipid peroxidation, and Ferrostatin-1 (Fer-1), a lipophilic antioxidative agent, successfully inhibited the increase in lipid peroxidation. Fer-1 suppressed the formation of EMT-associated fibrotic deposits, while EMT induction or Fer-1 treatment did not influence the cell viability or proliferation. Functionally, Fer-1 impeded EMT-driven cell migration and reduction in transepithelial electrical resistance. It demonstrated regulatory prowess by downregulating the mesenchymal marker fibronectin, upregulating the epithelial marker ZO-1, and inhibiting the EMT-associated transcriptional factor ZEB1. Additionally, VEGF, a major pathogenic cytokine in various retinal diseases, is also upregulated during EMT, and Fer-1 significantly mitigated the effect. The present study disclosed the involvement of lipid peroxidation in EMT of RPE cells, and suggests the suppression of lipid peroxidation may be a potential therapeutic target in retinal diseases in which EMT is implicated.

Disease-specific T cell receptors maintain pathogenic T helper cell responses in postinfectious Lyme arthritis.

Antibiotic-Refractory Lyme Arthritis (ARLA) involves a complex interplay of T cell responses targeting Borrelia burgdorferi antigens succeeding towards autoantigens by epitope spreading. However, the precise molecular mechanisms driving the pathogenic T cell response in ARLA remain unclear. Our aim was to elucidate the molecular program of disease-specific Th cells. Using flow cytometry, high-throughput T cell receptor (TCR) sequencing and scRNA-seq of CD4+ Th cells isolated from the joints of European ARLA patients, we aimed at inferring antigen specificity through unbiased analysis of TCR repertoire patterns, identifying surrogate markers for disease-specific TCRs and connecting TCR specificity to transcriptional patterns. PD-1hiHLA-DR+CD4+ effector T cells were clonally expanded within the inflamed joints and persisted throughout disease course. Among these cells, we identified a distinct TCRβ motif restricted to HLA-DRB1*11 or *13 alleles. These alleles, being underrepresented in North American ARLA patients, were unexpectedly prevalent in our European cohort. The identified TCRβ motif served as surrogate marker for a convergent TCR response specific to ARLA, distinguishing it from other rheumatic diseases. In the scRNA-seq dataset, the TCRβ motif particularly mapped to peripheral T helper (TPH) cells displaying signs of sustained proliferation, continuous TCR signaling, and expressing CXCL13 and IFN-γ. By inferring disease-specific TCRs from synovial T cells we identified a convergent TCR response in the joints of ARLA patients that continuously fueled the expansion of TPH cells expressing a pathogenic cytokine effector program. The identified TCRs will aid in uncovering the major antigen targets of the maladaptive immune response. Supported by the German Research Foundation (DFG) MO 2160/4-1; the Federal Ministry of Education and Research (BMBF; Advanced Clinician Scientist-Program INTERACT; 01EO2108) embedded in the Interdisciplinary Center for Clinical Research (IZKF) of the University Hospital Würzburg; the German Center for Infection Research (DZIF; Clinical Leave Program; TI07.001_007) and the Interdisciplinary Center for Clinical Research (IZKF) Würzburg (Clinician Scientist Program, Z-2/CSP-30).

Selective production of IL-33-neutralizing autoantibody ameliorates asthma responses and severity

BackgroundNatural anti-cytokine autoantibodies can regulate homeostasis of infectious and inflammatory diseases. The anti-cytokine autoantibody profile and relevance to the pathogenesis of asthma are unknown. We aim to identify key anti-cytokine autoantibodies in asthma patients, and reveal their immunological function and clinical significance. MethodsA Luciferase Immunoprecipitation System was used to screen serum autoantibodies against 11 key cytokines in patients with allergic asthma and healthy donors. The antigen-specificity, immunomodulatory functions and clinical significance of anti-cytokine autoantibodies were determined by ELISA, qPCR, neutralization assays and statistical analysis, respectively. Potential conditions for autoantibody induction were revealed by in vitro immunization. ResultsOf 11 cytokines tested, only anti-IL-33 autoantibody was significantly increased in asthma, compare to healthy controls, and the proportion positive was higher in patients with mild-to-moderate than severe allergic asthma. In allergic asthma patients, the anti-IL-33 autoantibody level correlated negatively with serum concentration of pathogenic cytokines (e.g., IL-4, IL-13, IL-25 and IL-33), IgE, and blood eosinophil count, but positively with mid-expiratory flow FEF25–75%. The autoantibodies were predominantly IgG isotype, polyclonal and could neutralize IL-33-induced pathogenic responses in vitro and in vivo. The induction of the anti-IL-33 autoantibody in blood B-cells in vitro required peptide IL-33 antigen along with a stimulation cocktail of TLR9 agonist and cytokines IL-2, IL-4 or IL-21. ConclusionsSerum natural anti-IL-33 autoantibodies are selectively induced in some asthma patients. They ameliorate key asthma inflammatory responses, and may improve lung function of allergic asthma.

CD4+ T Cell Effector Status Is Dependent on Aryl Hydrocarbon Receptor Activation during High Salt Diet

Recent studies suggest that patients with hypertension and individuals that consume a HSD are depleted of numerous tryptophan-derived indoles and other AHR-binding metabolites that are key in intestinal CD4+ T cell homeostasis. Thus, the inappropriate immunologic response by CD4+ T cells, and their role in hypertension, may be due in part to loss of AHR activation. Therefore, we hypothesized that AHR activation contributes to maintaining effector T cell status in salt-sensitive mice. To examine the influence of a representative homeostatic or proinflammatory environment and the subsequent response to salt, we cultured naïve CD4+ T cells under homeostatic cytokine or pathogenic cytokine conditions and exposed the cultures to increased NaCl conditions with or without the AHR-activating metabolite, FICZ (6-Formylindolo(3,2-b)-carbazole). TH17 cells cultured under homeostatic high salt conditions were depressed with a concomitant increase in response to FICZ. In contrast, TH17 cells cultured under pathogenic high salt conditions had augmented IL-17A-expressing cells without a response to AHR activation. These data suggest that excess salt is disrupting the ability to induce IL-17A and increasing AHR activity overcomes this disruption to promote IL-17A, without expansion of the pathogenic TH17 pool, and this observation was specific to non-pathogenic microenvironments (homeostatic TH17: media: 11±0.6%, +NaCl: 4.9±0.3%*, +NaCl/FICZ: 9.9±0.8%, of CD4; pathogenic TH17: media: 1.0±0.2%, +NaCl: 2.2±0.2%*, +NaCl/FICZ: 2.2±0.2%*, *vs respective media, n=8/group). Given these data, we then fed salt-sensitive mice (nitric oxide synthase inhibition) a HSD or a HSD supplemented with the AHR agonist indole-3-carbinol (I3C) and examined Peyer’s patches, a lymphoid tissue in close proximity to intestinal lumen contents enriched with salt and the I3C-metabolites and noted expansion of non-pathogenic TH17 cells. In conclusion, increasing AHR ligand availability through supplementing I3C may provide a gut-centric experimental approach in targeting experimental salt-sensitivity as it relates to immunologic disturbances. F31HL151264. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Targeting purinergic signaling in platelets ameliorates the bacterial burden and reduces the cytokine storm in the peritoneal cavity during intra-abdominal sepsis

Sepsis is a complex clinical syndrome of the host response to an infection, involving a pathogenic cytokine storm that drives inflammatory injury in multiple organ systems. The abdomen is a major site of sepsis, contributing to high mortality. Platelets express the purinergic receptors P2Y1 and P2Y12; once activated, these receptors mediate the secretion of pro-inflammatory mediators that contribute to organ damage in sepsis. Systemic blockade of P2Y1 or P2Y12 decreased organ damage and inflammatory cytokine levels in a sex-related manner during sepsis. Here, we hypothesize that blocking P2Y1 or P2Y12 activity enhances pathogen clearance in the peritoneal cavity during intra-abdominal sepsis in a sex-specific manner. Male and female C57BL6/6J mice underwent sham surgery or cecal ligation and puncture (CLP) to induce sepsis. P2Y1 or P2Y12 antagonists, MRS2279 (10 mg/kg) and Ticagrelor (30 mg/kg) respectively, were administered intraperitoneally after surgery. The peritoneal cavity fluid (PCF) was collected 24 hours post-surgery by peritoneal lavage. To assess cell recruitment in the cavity, we measured leukocyte and platelet counts using Hemavet® Multispecies Hematology System. To assess inflammation, we measured PCF levels of the cytokines MIP-1α and Rantes as well as bacterial clearance. The ileum of the small intestinal tissue samples were collected 24 hours post-surgery and platelet infiltration was measured by platelet factor 4 (PF4) content in the tissue using an ELISA kit. In response to the P2Y1 or P2Y12 blocker, we noted a significant reduction in the bacteria content in the PCF of CLP mice. Blocking P2Y12 but not P2Y1 reduced leukocyte recruitment, while platelet counts were reduced only with P2Y1 blockade in septic males. The sepsis-induced increase in MIP-1α levels was not noted upon P2Y1 but not P2Y12 blockage in the PCF of septic males. However, Rantes levels in the PCF were reduced for either treatment in males compared to the untreated CLP. The platelet marker PF4 in the ileum was increased during sepsis but reduced after either treatment in sepsis males as compared to the untreated CLP. In female CLP mice, a significant reduction in bacterial burden in the PCF in response to the P2Y1 but not the P2Y12 blocker was shown. Blocking P2Y1 or P2Y12 reduced leukocyte recruitment and platelet counts in female CLP mice. As observed in males, PF4 was increased in the ileum during sepsis but reduced following exposure to either treatment in females. No change was noted for MIP-1α or Rantes levels in the females among all the septic groups. Our data indicate that blocking purinergic signaling and in particular P2Y12 in the peritoneal cavity in males prevents bacterial spreading and diminishes cytokine levels, indicating that it could be an effective treatment for intra-abdominal sepsis. However, in female mice blocking the receptor P2Y1, but not P2Y12 has shown to be optimal. Targeting purinergic signaling is a promising sex-specific therapeutic strategy for sepsis. This work was supported by the Research grant AI156627-01 (EL) from the National Institute of Health. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A Mendelian randomization study on the causal effects of circulating cytokines on the risk of vitiligo.

Accumulating evidence reveals an association between circulating cytokine levels and vitiligo. However, the causal association between circulating cytokine levels and vitiligo remains unrevealed. We performed a two-sample Mendelian randomization (MR) analysis using a genome-wide association study of the 41 cytokines dataset, which was conducted with 3 Finnish cohorts (n = 8,293). Vitiligo data were acquired from strictly defined vitiligo data collected by FinnGenbiobank analysis, which included 207,613 European ancestors (131 vitiligo patients, 207,482 controls). The inverse-variance weighted (IVW) method, weighted median (WME), simple model, weighted model, and MR-Egger were used to determine the changes in vitiligo pathogenic cytokine taxa, followed by sensitivity analysis, including horizontal pleiotropy analysis. The MR Steiger test evaluated the strength of a causal association, and the leave-one-out method was used to assess the reliability of the results. The possibility of reverse causality was also investigated using a reverse MR study. We observed that rising IL-4 levels generated an enhanced probability of vitiligo in IVW (OR 2.72, 95%CI 1.19-6.22, p = 0.018). According to the results of the MR analysis, there were causal links between IL-4 and vitiligo. Results were steady after sensitivity and heterogeneity analyses. Our research reveals that a genetically determined increased level of circulating IL-4 may be linked to a higher risk of developing vitiligo. The development of innovative treatment approaches (such as tofacitinib or dupilumab) that focus on blocking IL-4 as a novel way of preventing and treating vitiligo is significantly impacted by our findings.

Interleukin-15 is a hair follicle immune privilege guardian

The autoimmunity-promoting cytokine, Interleukin-15 (IL-15), is often claimed to be a key pathogenic cytokine in alopecia areata (AA). Yet, rhIL-15 promotes human hair follicle (HF) growth ex vivo. We have asked whether the expression of IL-15 and its receptor (IL-15R) isoforms is altered in human AA and how IL-15 impacts on human HF immune privilege (HF-IP) in the presence/absence of interferon-γ (IFNγ), the well-documented key AA-pathogenic cytokine, as well as on hair regrowth after experimental AA induction in vivo. Quantitative immunohistomorphometry showed the number of perifollicular IL-15+ T cells in AA skin biopsies to be significantly increased compared to healthy control skin, while IL-15, IL-15Rα, and IL-15Rγ protein expression within the hair bulb were significantly down-regulated in AA HFs. In organ-cultured human scalp HFs, rhIL-15 significantly reduced hair bulb expression of MICA, the key “danger” signal in AA pathogenesis, and increased production of the HF-IP guardian, α-MSH. Crucially, ex vivo, rhIL-15 prevented IFNγ-induced HF-IP collapse, restored a collapsed HF-IP by IL-15Rα-dependent signaling (as documented by IL-15Rα−silencing), and protected AA-preventive immunoinhibitory iNKT10 cells from IFNγ-induced apoptosis. rhIL-15 even promoted hair regrowth after experimental AA induction in human scalp skin xenotransplants on SCID/beige mice in vivo. Our data introduce IL-15 as a novel, functionally important HF-IP guardian whose signaling is constitutively defective in scalp HFs of AA patients. Our data suggest that selective stimulation of intrafollicular IL-15Rα signaling could become a novel therapeutic approach in AA management, while blocking it pharmacologically may hinder both HF-IP restoration and hair re-growth and may thus make HFs more vulnerable to AA relapse.

The Therapeutic Potential of Copper Peptides on Atopic Dermatitis Lesions in BALB/c mice and Human Keratinocytes

PURPOSE: Atopic dermatitis (AD), often referred to as eczema, is a chronic disease characterized by skin inflammation, itchiness, irritation, and dryness. It is caused mainly by immune dysregulation. Copper peptides (CP) have been used for a variety of skin-related purposes. In this study, we examined the effect of CP on AD induced in BALB/c mice.METHODS: BALB/c mice were separated into the following five groups: control, AD-only, AD+CP10, AD+CP100, and AD+CERA. AD was induced using house dust mite (HDM) extract and 2,4-dinitrochlorobenzene (DNCB). The mice received CP or ceramide (CERA; positive control) based on their group allocation. Histopathological analysis was performed to assess the effect of CP on mast cell infiltration. Dermal and epidermal thickness were measured using microphotographs of hematoxylin-and-eosin-stained tissue. We examined the serum immunoglobulin E (IgE) levels and mRNA gene expression of pathogenic cytokines in the mouse ear tissue and in human keratinocytes.RESULTS: CP treatment decreased dermal and epidermal mouse ear thickness and mast cell infiltration in the ear tissue in a dosedependent manner. It also decreased the serum IgE levels and the mRNA gene expression of pathogenic cytokines in the ear tissue and in the human keratinocytes. Interestingly, the therapeutic effect of CP was more robust than that of CERA.CONCLUSIONS: These results indicate that CP may have therapeutic potential against AD.

Th17 cell promotes apoptosis of IL-23R+ neurons in experimental autoimmune encephalomyelitis

Myelin antigen-reactive Th1 and Th17 cells are critical drivers of central nervous system (CNS) autoimmune inflammation. Transcription factors T-bet and RORγt play a crucial role in the differentiation and function of Th1 and Th17 cells, and impart them a pathogenic role in CNS autoimmune inflammation. Mice deficient in these two factors do not develop experimental autoimmune encephalomyelitis (EAE). While T-bet and RORγt are known to regulate the expression of several cell adhesion and migratory molecules in T cells, their role in supporting Th1 and Th17 trafficking to the CNS is not completely understood. More importantly, once Th1 and Th17 cells reach the CNS, how the function of these transcription factors modulates the local inflammatory response during EAE is unclear. In the present study, we showed that myelin oligodendrocyte glycoprotein 35–55 peptide (MOG35–55)-specific Th1 cells deficient in RORγt could cross the blood-brain barrier (BBB) but failed to induce demyelination, apoptosis of neurons, and EAE. Pathogenic Th17 cell-derived cytokines GM-CSF, TNF-α, IL-17A, and IL-21 significantly increased the surface expression of IL-23R on neuronal cells. Furthermore, we showed that, in EAE, neurons in the brain and spinal cord express IL-23R. IL-23-IL-23R signaling in neuronal cells caused phosphorylation of STAT3 (Ser727 and Tyr705) and induced cleaved caspase 3 and cleaved poly (ADP-ribose) polymerase-1 (PARP-1) molecules in an IL-23R-dependent manner and caused apoptosis. Thus, we provided a mechanism showing that T-bet is required to recruit pathogenic Th17 cells to the CNS and RORγt-mediated inflammatory response to drive the apoptosis of IL-23R+ neurons in the CNS and cause EAE. Understanding detailed molecular mechanisms will help to design better strategies to control neuroinflammation and autoimmunity. One Sentence SummaryIL-23-IL-23R signaling promotes apoptosis of CNS neurons.

A unique profile of serum cytokines in type 1 autoimmune pancreatitis and chronic rhinosinusitis.

Type 1 autoimmune pancreatitis (AIP) is a pancreatic manifestation of IgG4-related disease (IgG4-RD). Although AIP and IgG4-RD are characterized by multiple organ involvement including salivary glands, lung, and kidney, co-occurrence of chronic rhinosinusitis (CRS) and AIP/IgG4-RD has been poorly defined. We explored molecular mechanism accounting for the co-occurrence of CRS and AIP/IgG4-RD. Serum concentrations of IFN-α and IL-33 were measured by enzyme-linked immune-sorbent assay. We encountered a patient with concurrent type 1 AIP/IgG4-RD and CRS. Induction of remission by prednisolone (PSL) for type 1 AIP/IgG4-RD led to a marked improvement of CRS. Serum cytokine analysis after PSL treatment revealed a marked reduction in serum concentrations of IFN-α and IL-33, both of which are candidate pathogenic cytokines for AIP/IgG4-RD. Given that IL-33 is shared as one of pathogenic cytokines by type 1 AIP/IgG4-RD and CRS, enhanced IL33 responses may cause concurrent type 1 AIP/IgG4-RD and CRS.

Exosomal miRNAs in autoimmune skin diseases.

Exosomes, bilaterally phospholipid-coated small vesicles, are produced and released by nearly all cells, which comprise diverse biological macromolecules, including proteins, DNA, RNA, and others, that participate in the regulation of their biological functions. An increasing number of studies have revealed that the contents of exosomes, particularly microRNA(miRNA), play a significant role in the pathogenesis of various diseases, including autoimmune skin diseases. MiRNA is a class of single-stranded non-coding RNA molecules that possess approximately 22 nucleotides in length with the capability of binding to the untranslated as well as coding regions of target mRNA to regulate gene expression precisely at the post-transcriptional level. Various exosomal miRNAs have been found to be significantly expressed in some autoimmune skin diseases and involved in the pathogenesis of conditions via regulating the secretion of crucial pathogenic cytokines and the direction of immune cell differentiation. Thus, exosomal miRNAs might be promising biomarkers for monitoring disease progression, relapse and reflection to treatment based on their functions and changes. This review summarized the current studies on exosomal miRNAs in several common autoimmune skin diseases, aiming to dissect the underlying mechanism from a new perspective, seek novel biomarkers for disease monitoring and lay the foundation for developing innovative target therapy in the future.

IL-17A Orchestrates Reactive Oxygen Species/HIF1α-Mediated Metabolic Reprogramming in Psoriasis.

Immune cell-derived IL-17A is one of the key pathogenic cytokines in psoriasis, an immunometabolic disorder. Although IL-17A is an established regulator of cutaneous immune cell biology, its functional and metabolic effects on nonimmune cells of the skin, particularly keratinocytes, have not been comprehensively explored. Using multiomics profiling and systems biology-based approaches, we systematically uncover significant roles for IL-17A in the metabolic reprogramming of human primary keratinocytes (HPKs). High-throughput liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance spectroscopy revealed IL-17A-dependent regulation of multiple HPK proteins and metabolites of carbohydrate and lipid metabolism. Systems-level MitoCore modeling using flux-balance analysis identified IL-17A-mediated increases in HPK glycolysis, glutaminolysis, and lipid uptake, which were validated using biochemical cell-based assays and stable isotope-resolved metabolomics. IL-17A treatment triggered downstream mitochondrial reactive oxygen species and HIF1α expression and resultant HPK proliferation, consistent with the observed elevation of these downstream effectors in the epidermis of patients with psoriasis. Pharmacological inhibition of HIF1α or reactive oxygen species reversed IL-17A-mediated glycolysis, glutaminolysis, lipid uptake, and HPK hyperproliferation. These results identify keratinocytes as important target cells of IL-17A and reveal its involvement in multiple downstream metabolic reprogramming pathways in human skin.

Preclinical Evaluation of INCB160058 - a Novel and Potentially Disease-Modifying Therapy for JAK2V617F Mutant Myeloproliferative Neoplasms

The Janus kinase 2 ( JAK2) mutation, JAK2V617F, is the most common oncogenic driver in myeloproliferative neoplasms (MPNs), with nearly all cases of polycythemia vera (PV) and over half of primary myelofibrosis (MF) and essential thrombocythemia (ET) patients positive for the somatic mutation. Approved therapies for MPNs such as ruxolitinib, which act by directly inhibiting activity of the kinase domain (JH1) of JAK2, have demonstrated impressive clinical efficacy and safety in patients with MPNs; however, they do not address JAK2V617F allelic burden or achieve molecular remission of disease. A targeted JAK2V617F selective agent sparing wild-type (WT) JAK2 activity has potential to eliminate mutant cells, induce molecular remission, and theoretically lead to functional cure of MPNs. We report herein the preclinical development of pseudokinase (JH2)-targeting INCB160058, a first-in-class, orally bioavailable small molecule with the ability to selectively target JAK2V617F + cell populations derived from patients with JAK-mutant MPNs. Using structure- and function-guided molecular design, INCB160058 was designed to bind with picomolar affinity to the JH2 domain of JAK2V617F at the canonical ATP-binding site, with high specificity (>2500-fold) relative to binding at the active kinase domain (JH1) targeted by currently approved JAK inhibitors. Live cell single-molecule fluorescence microscopy showed that INCB160058 binding to JAK2V617F blocked ligand-independent thrombopoietin receptor dimerization induced by the mutation, and consequently led to loss of JH1 domain kinase activity. X-ray crystallography analysis indicates that the observed inhibition is likely driven by conformational disruption of the ⍺C helix motif at Phe 594 and Phe 595 in conjunction with a shift of the upstream region from Leu 583 to Asn 589 upon INCB160058 binding to the JH2 domain of JAK2V617F. We utilized both CD34 + human multipotent hematopoietic stem cells derived from patients with JAK2-mutant MF, engineered JAK2-mutant human hematopoietic cancer cell lines (eg, SET2 and UKE-1), and murine BA/F3 cell lines to explore the selective effects of INCB160058 on JAK2V617F compared with WT JAK2. INCB160058 treatment selectively reduced pathogenic phospho-STAT5 levels, decreased abnormal megakaryopoiesis, and suppressed colony formation only in JAK2V617F + CD34 + cells but not in CD34 + cells from healthy volunteers. Importantly, continuous exposure of mutant and WT JAK2 cells to INCB160058 in co-cultures at concentrations below IC 50 resulted in progressive elimination of JAK2V617F + cells without affecting WT cells. At the end of the testing period, the JAK2V617F-harboring population was no longer detectable in the co-culture assay. In NSG mice subcutaneously inoculated with JAK2V617F-expressing SET2 cells, INCB160058 was tolerated and exhibited significant antitumor activity. In addition, following INCB160058 treatment, a significant reduction in the engraftment of total human cells, particularly human erythroid progenitors (hCD45 − mCD45 − Ter119 − hCD71 + hCD235a +), was observed in NSGS mice xenotransplanted with JAK2V617F + CD34 + cells. Moreover, INCB160058 treatment also led to the normalization of various pathogenic cytokines, such as interleukin (IL)-6 and IL-8. Importantly, these observations were absent in NSGS mice engrafted with CD34 + cells from healthy volunteers following INCB160058 treatment, further demonstrating the selectivity of INCB160058 for JAK2V617F. In summary, our results indicate a novel mechanism of action of INCB160058, a high-affinity pseudokinase (JH2) binding inhibitor of JAK2V617F that blocks cytokine-independent activity of JAK2V617F while preserving cytokine-dependent signaling. Extended treatment with INCB160058 at low therapeutic doses results in the specific elimination of mutant JAK2V617F-harboring cells in mouse models and human cancer cells with minimal impact on WT counterparts. Clinical testing of INCB160058 may allow patients with MPNs to achieve molecular remission by eliminating cells with the main genetic aberration and afford an opportunity to overcome the disease.

Anti-prolactin treatment alleviates lupus conditions by regulating the JAK2-STAT3 pathway.

We previously revealed the role of prolactin (PRL) in antibody production and disease activity in patients with systemic lupus erythematosus. In this study, we sought to determine whether inhibition of PRL could improve lupus-like disease in MRL/lpr mice. The expression levels of PRL in various cell types of lupus patients were measured by flow cytometry. The effects of anti-PRL on animal survival, renal histopathology, creatinine, proteinuria, anti-dsDNA antibody, cytokine production, splenomegaly and lymphadenopathy were assessed. The effect of anti-PRL on the Jak2-Stat3 signalling pathway was detected by western blotting. Prolactin was upregulated in B cells, neutrophils, CD4+ T cells, and monocytes isolated from patients with lupus. Furthermore, inhibition of PRL by anti-PRL treatment around the time of onset prolonged the survival of MRL/lpr mice, significantly reduced anti-dsDNA antibody production, and alleviated symptoms of lupus nephritis, splenomegaly, and lymphadenopathy. In addition, anti-PRL-treated mice showed a decrease in the levels of pathogenic cytokines such as IL-21 and IL-6. Furthermore, mechanistically, anti-PRL treatment significantly reduced the levels of p-Jak2 and p-Stat3 in MRL/lpr mice. In summary, these data suggest that PRL inhibition alleviates lupus-like disease in MRL/lpr mice by modulating the Jak2-Stat3 signalling cascade. More importantly, our results imply the potential of PRL inhibitors and may provide a novel therapeutic approach for lupus.

ISG15–USP18 Dysregulation by Oxidative Stress Promotes IFN-γ Secretion from CD8+ T Cells in Vitiligo

Excessive oxidative stress is thought to play pathologic roles in cellular senescence and autoimmune disorders by inducing inflammation and breaking down immune tolerance. In this study, we sought to identify the factors linking oxidative stress to autoimmunity and cellular senescence in vitiligo, where elevated oxidative stress plays an important role. RNA sequencing analysis of hydrogen peroxide-treated melanocytes revealed upregulation of ISG15. The upregulation of ISG15 was observed in vitiligo skin tissues as well as in the blood of patients with vitiligo, whereas USP18 downregulation was observed in vitiligo melanocytes and vitiligo skin tissues. Oxidative stress induced hypermethylation of the USP18 promoter region in keratinocytes and melanocytes, and USP18 promoter hypermethylation was also confirmed in vitiligo skin tissues. Our results indicate that USP18 promoter hypermethylation caused by oxidative stress increases ISG15 expression in keratinocytes and melanocytes along with senescence changes, leading CD8+ T cells to produce IFN-γ, the main pathogenic cytokine in vitiligo. Therefore, the ISG15-USP18 network may be important in oxidative stress-induced autoimmunity and cellular senescence in vitiligo pathogenesis.

Calcipotriene and Betamethasone Dipropionate PAD-Cream Demonstrates Greater Treatment Efficacy in Patients with Moderate-to-Severe Psoriasis Compared to Topical Suspension/Gel: A Subgroup Analysis of Two Phase3 Studies.

Psoriasis ranges from mild to severe with the majority of patients having mild disease. Mild to moderate disease is often treated with topical therapies while photo-, oral, and biologic therapies are generally reserved for moderate-to-severe disease. There is a strong scientific rationale for the combination of calcipotriene (CAL) and betamethasone dipropionate (BDP) with respect to mode of action, efficacy, and safety and CAL/BDP has shown an inhibitory effect on key pathogenic cytokines in psoriasis including tumor necrosis factor-α, interleukin (IL)-17, and IL-23. The objective of this pooled posthoc analysis is to investigate the efficacy of CAL/BDP polyaphron dispersion (PAD)-cream in subgroups of patients with moderate-to-severe psoriasis from two completed phase3 studies conducted in the USA and Europe. The proportion of patients achieving Physician Global Assessment (PGA) treatment success as well as a modified Psoriasis Area and Severity Index (mPASI)75 response was higher in the subgroup with a body surface area > 10% and mPASI > 10 and Dermatology Life Quality Index > 10 at baseline compared to the overall patient population. Furthermore, the numerical difference in treatment efficacy between CAL/BDP PAD-cream and CAL/BDP topical suspension/gel increased in patient subgroups with higher baseline severity. Similar patterns were shown for the patient-reported outcomes. In this subgroup analysis, patients who had higher disease severity at baseline achieved greater efficacy than the total patient population when treated with 8weeks of CAL/BDP PAD-cream as compared to a currently marketed active comparator. Additionally, as indicated by this analysis, CAL/BDP PAD-cream treatment may also be more convenient and less greasy, which may reduce the burden of daily treatment and improve adherence to therapy. NCT03308799 and NCT03802344.

Single-Cell transcriptomes of immune cells provide insights into the therapeutic effects of mycophenolate mofetil on autoimmune uveitis.

Mycophenolate mofetil (MMF) is an immunosuppressive agent widely applied in various autoimmune diseases, including autoimmune uveitis, a sight-threatening autoimmune disease mainly affecting the eyes. However, the mechanisms of action are not comprehensively understood. To investigate the potential impact of MMF on uveitis, we generated single-cell RNA sequence data from normal, experimental autoimmune uveitis (EAU) and MMF-treated EAU mice. We observed that some EAU-induced transcriptional changes were reversed by MMF treatment. Transcriptional data indicated that MMF may have a general inhibitory effect on the activation of immune cells during EAU. Each immune cell type showed a different response to MMF treatment. Pseudotime analysis showed that MMF treatment partly reversed the increased differentiation tendency from naïve to effector phenotypes of T and B cells in EAU. The reduced proportion of T-helper (Th)1 and T-helper (Th)17 cells after MMF treatment was confirmed using flow cytometry. MMF treatment downregulated the EAU-associated upregulation of several molecules (such as Cebpd, Pim1, Furin, Bhlhe40, and Hif1a) that promote pathogenic cytokine production by T helper (Th)-1 and Th17 cells. Abnormally enhanced immunoglobulin production, antigen processing, and presentation ability of B cells may also be inhibited by MMF treatment. In addition to T and B cells, MMF treatment countered EAU-induced transcriptional changes in other immune cells to different degrees. Overall, our findings provide novel insights into the mechanisms underlying MMF treatment and indicate that the therapeutic effect of MMF is not driven by a single molecule.