Selective Janus kinase 1 inhibition resolves inflammation and restores hair growth offering a viable treatment option for alopecia areata.

Background: Janus Kinase (JAK) inhibition has recently demonstrated therapeutic efficacy in both restoring hair growth and resolving inflammation in Alopecia Areata (AA). These effects are dose dependent and mainly efficacious at ranges close to a questionable risk profile. Objectives: We explored the possibility to separate the beneficial and adverse effects of JAK inhibition by selectively inhibiting JAK1 and thereby avoiding side effects associated with JAK2 blockade. Methods: The C3H/HeJ mouse model of AA was used to demonstrate therapeutic efficacy in vivo with different regimens of a selection of JAK inhibitors in regards to systemic versus local drug exposure. Human peripheral blood lymphocytes were stimulated in vitro to demonstrate translation to the human situation. Results: We demonstrate that selective inhibition of JAK1 produces fast resolution of inflammation and complete restoration of hair growth in the C3H/HeJ mouse model of AA. Furthermore, we show that topical treatment does not restore hair growth and that treatment needs to be extended well beyond that of restored hair growth in order to reach treatment-free remission. For translatability to human disease, we show that cytokines involved in AA pathogenesis are similarly inhibited by selective JAK1 and pan-JAK inhibition in stimulated human peripheral lymphocytes and specifically in CD8+ T cells. Conclusion: This study demonstrates that systemic exposure is required for efficacy in AA and we propose that a selective JAK1 inhibitor will offer a treatment option with a superior safety profile to pan-JAK inhibitors for these patients.

Deep Learning With Conformal Prediction for Hierarchical Analysis of Large-Scale Whole-Slide Tissue Images.

With the increasing amount of image data collected from biomedical experiments there is an urgent need for smarter and more effective analysis methods. Many scientific questions require analysis of image sub-regions related to some specific biology. Finding such regions of interest (ROIs) at low resolution and limiting the data subjected to final quantification at full resolution can reduce computational requirements and save time. In this paper we propose a three-step pipeline: First, bounding boxes for ROIs are located at low resolution. Next, ROIs are subjected to semantic segmentation into sub-regions at mid-resolution. We also estimate the confidence of the segmented sub-regions. Finally, quantitative measurements are extracted at full resolution. We use deep learning for the first two steps in the pipeline and conformal prediction for confidence assessment. We show that limiting final quantitative analysis to sub-regions with full confidence reduces noise and increases separability of observed biological effects.

Human Lung Conventional Dendritic Cells Orchestrate Lymphoid Neogenesis during Chronic Obstructive Pulmonary Disease.

Rationale: Emerging evidence supports a crucial role for tertiary lymphoid organs (TLOs) in chronic obstructive pulmonary disease (COPD) progression. However, mechanisms of immune cell activation leading to TLOs in COPD remain to be defined.Objectives: To examine the role of lung dendritic cells (DCs) in T follicular helper (Tfh)-cell induction, a T-cell subset critically implicated in lymphoid organ formation, in COPD.Methods: Myeloid cell heterogeneity and phenotype were studied in an unbiased manner via single-cell RNA sequencing on HLA-DR+ cells sorted from human lungs. We measured the in vitro capability of control and COPD lung DC subsets, sorted using a fluorescence-activated cell sorter, to polarize IL-21+CXCL13+ (IL-21-positive and C-X-C chemokine ligand type 13-positive) Tfh-like cells. In situ imaging analysis was performed on Global Initiative for Chronic Obstructive Lung Disease stage IV COPD lungs with TLOs.Measurements and Main Results: Single-cell RNA-sequencing analysis revealed a high degree of heterogeneity among human lung myeloid cells. Among these, conventional dendritic type 2 cells (cDC2s) showed increased induction of IL-21+CXCL13+ Tfh-like cells. Importantly, the capacity to induce IL-21+ Tfh-like cells was higher in cDC2s from patients with COPD than in those from control patients. Increased Tfh-cell induction by COPD cDC2s correlated with increased presence of Tfh-like cells in COPD lungs as compared with those in control lungs, and cDC2s colocalized with Tfh-like cells in TLOs of COPD lungs. Mechanistically, cDC2s exhibited a unique migratory signature and (transcriptional) expression of several pathways and genes related to DC-induced Tfh-cell priming. Importantly, blocking the costimulatory OX40L (OX40 ligand)-OX40 axis reduced Tfh-cell induction by control lung cDC2s.Conclusions: In COPD lungs, we found lung EBI2+ (Epstein-Barr virus-induced gene 2-positive) OX-40L-expressing cDC2s that induced IL-21+ Tfh-like cells, suggesting an involvement of these cells in TLO formation.

Dual Role For A MEK Inhibitor As A Modulator Of Inflammation And Host Defense Mechanisms With Potential Therapeutic Application In COPD.

Background: Unlike p38 mitogen-activated protein Kinases (MAPK) that has been extensively studied in the context of lung-associated pathologies in COPD, the role of the dual-specificity mitogen-activated protein kinase kinase (MEK1/2) or its downstream signaling molecule extracellular signal-regulated kinases 1/2 (ERK1/2) in COPD is poorly understood. Objectives: The aim of this study was to address whether MEK1/2 pathway activation is linked to COPD and that targeting this pathway can improve lung inflammation through decreased immune-mediated inflammatory responses without compromising bacterial clearance. Methods: Association of MEK1/2 pathway activation to COPD was investigated by immunohistochemistry using lung tissue biopsies from COPD and healthy individuals and through analysis of sputum gene expression data from COPD patients. The anti-inflammatory effect of MEK1/2 inhibition was assessed on cytokine release from lipopolysaccharide-stimulated alveolar macrophages. The effect of MEK1/2 inhibition on bacterial clearance was assessed using Staphylococcus aureus killing assays with RAW 264.7 macrophage cell line and human neutrophils. Results: We report here MEK1/2 pathway activation demonstrated by increased pERK1/2 staining in bronchial epithelium and by the presence of MEK gene activation signature in sputum samples from COPD patients. Inhibition of MEK1/2 resulted in a superior anti-inflammatory effect in human alveolar macrophages in comparison to a p38 inhibitor. Furthermore, MEK1/2 inhibition led to an increase in bacterial killing in human neutrophils and RAW 264.7 cells that was not observed with the p38 inhibitor. Conclusion: Our data demonstrate the activation of MEK1/2 pathway in COPD and highlight a dual function of MEK1/2 inhibition in improving host defense responses whilst also controlling inflammation.

Mechanisms of tolerance and potential therapeutic interventions in Alopecia Areata.

This review aims to address the mechanisms of compromised immune tolerance contributing to the development and maintenance of Alopecia Areata (AA). Our goal is to also highlight future treatment opportunities and therapeutics that will safely and efficiently restore hair growth and maintain patients in remission. AA is a presumptive autoimmune disorder that coincides and genetically clusters to several other autoimmune diseases. In this review, we pay attention to the learnings from the mechanistic research and drug development in these other autoimmune conditions. Interestingly, most of these diseases have been linked to compromised central and peripheral tolerance, and increased intestinal inflammation with enhanced gut permeability. Break of tolerance and priming of the autoreactive T-cells to attack antigenic epitopes in the hair follicle most likely requires several steps which include escape from negative selection and compromised peripheral tolerance. Local skin-related changes are also of importance due to the patchy manifestation of the skin areas with loss of hair, particularly in the early disease. Here, we discuss the defective mechanisms of tolerance, both central and peripheral, and hypothesize that the disease is driven by areas of tolerance break, and that these could be targeted for successful therapeutic interventions.

Thymic exosomes promote the final maturation of thymocytes.

Extensive knowledge has been gained the last years concerning mechanisms underlying the selection of single positive thymocytes in the thymic medulla. Less is known regarding other important processes in the thymic medulla such as the regulation of late stage thymocyte maturation. We have previously reported that exosomes are abundant in the thymus with a phenotype that indicates an epithelial cell origin and immunoregulatory properties. In this study we use an in vitro system to investigate the effects of thymic exosomes on the maturation of single positive thymocytes as well as effects on nTreg formation. We show that thymic exosomes promote the maturation of single positive CD4+CD25- cells into mature thymocytes with S1P1+Qa2+ and CCR7+Qa2+ phenotypes. Furthermore, we show that thymic exosomes reduce the formation of CD4+CD25+FoxP3+ thymocytes and that these exosome effects are independent of dendritic cell co-stimulation but require intact exosomal RNA content and surface proteins. An efficient direct uptake of exosomes by both thymocytes and thymic DC's is also demonstrated. In conclusion, this study demonstrates that exosomes may represent a new route of communication within the thymus.

Delivery of Small Interfering RNAs to Cells via Exosomes.

Exosomes are small membrane bound vesicles between 30 and 100 nm in diameter of endocytic origin that are secreted into the extracellular environment by many different cell types. Exosomes play a role in intercellular communication by transferring proteins, lipids, and RNAs to recipient cells.Exosomes from human cells could be used as vectors to provide cells with therapeutic RNAs. Here we describe how exogenous small interfering RNAs may successfully be introduced into various kinds of human exosomes using electroporation and subsequently delivered to recipient cells. Methods used to confirm the presence of siRNA inside exosomes and cells are presented, such as flow cytometry, confocal microscopy, and Northern blot.

Exosomes in the Thymus: Antigen Transfer and Vesicles.

Thymocytes go through several steps of maturation and selection in the thymus in order to form a functional pool of effector T-cells and regulatory T-cells in the periphery. Close interactions between thymocytes, thymic epithelial cells, and dendritic cells are of vital importance for the maturation, selection, and lineage decision of the thymocytes. One important question that is still unanswered is how a relatively small epithelial cell population can present a vast array of self-antigens to the manifold larger population of developing thymocytes in this selection process. Here, we review and discuss the literature concerning antigen transfer from epithelial cells with a focus on exosomes. Exosomes are nano-sized vesicles released from a cell into the extracellular space. These vesicles can carry proteins, microRNAs, and mRNAs between cells and are thus able to participate in intercellular communication. Exosomes have been shown to be produced by thymic epithelial cells and to carry tissue-restricted antigens and MHC molecules, which may enable them to participate in the thymocyte selection process.

Human thymic epithelial primary cells produce exosomes carrying tissue-restricted antigens.

Exosomes are nano-sized vesicles released by cells into the extracellular space and have been shown to be present in thymic tissue both in mice and in humans. The source of thymic exosomes is however still an enigma and hence it is not known whether thymic epithelial cells (TECs) are able to produce exosomes. In this work, we have cultured human TECs and isolated exosomes. These exosomes carry tissue-restricted antigens (TRAs), for example, myelin basic protein and desmoglein 3. The presence of TRAs indicates a possible role for thymic epithelium-derived exosomes in the selection process of thymocytes. The key contribution of these exosomes could be to disseminate self-antigens from the thymic epithelia, thus making them more accessible to the pool of maturing thymocytes. This would increase the coverage of TRAs within the thymus, and facilitate the process of positive and negative selection.

Premature thymic involution is independent of structural plasticity of the thymic stroma.

The thymus is the organ devoted to T-cell production. The thymus undergoes multiple rounds of atrophy and redevelopment before degenerating with age in a process known as involution. This process is poorly understood, despite the influence the phenomenon has on peripheral T-cell numbers. Here we have investigated the FVB/N mouse strain, which displays premature thymic involution. We find multiple architectural and cellular features that precede thymic involution, including disruption of the epithelial-endothelial relationship and a progressive loss of pro-T cells. The architectural features, reminiscent of the human thymus, are intrinsic to the nonhematopoietic compartment and are neither necessary nor sufficient for thymic involution. By contrast, the loss of pro-T cells is intrinsic to the hematopoietic compartment, and is sufficient to drive premature involution. These results identify pro-T-cell loss as the main driver of premature thymic involution, and highlight the plasticity of the thymic stroma, capable of maintaining function across diverse interstrain architectures.

Altered expression of autoimmune regulator in infant down syndrome thymus, a possible contributor to an autoimmune phenotype.

Down syndrome (DS), caused by trisomy of chromosome 21, is associated with immunological dysfunctions such as increased frequency of infections and autoimmune diseases. Patients with DS share clinical features, such as autoimmune manifestations and specific autoantibodies, with patients affected by autoimmune polyendocrine syndrome type 1. Autoimmune polyendocrine syndrome type 1 is caused by mutations in the autoimmune regulator (AIRE) gene, located on chromosome 21, which regulates the expression of tissue-restricted Ags (TRAs) in thymic epithelial cells. We investigated the expression of AIRE and TRAs in DS and control thymic tissue using quantitative PCR. AIRE mRNA levels were elevated in thymic tissue from DS patients, and trends toward increased expression of the AIRE-controlled genes INSULIN and CHRNA1 were found. Immunohistochemical stainings showed altered cell composition and architecture of the thymic medulla in DS individuals with increased frequencies of AIRE-positive medullary epithelial cells and CD11c-positive dendritic cells as well as enlarged Hassall's corpuscles. In addition, we evaluated the proteomic profile of thymic exosomes in DS individuals and controls. DS exosomes carried a broader protein pool and also a larger pool of unique TRAs compared with control exosomes. In conclusion, the increased AIRE gene dose in DS could contribute to an autoimmune phenotype through multiple AIRE-mediated effects on homeostasis and function of thymic epithelial cells that affect thymic selection processes.

Characterization of human thymic exosomes.

Exosomes are nanosized membrane-bound vesicles that are released by various cell types and are capable of carrying proteins, lipids and RNAs which can be delivered to recipient cells. Exosomes play a role in intercellular communication and have been described to mediate immunologic information. In this article we report the first isolation and characterization of exosomes from human thymic tissue. Using electron microscopy, particle size determination, density gradient measurement, flow cytometry, proteomic analysis and microRNA profiling we describe the morphology, size, density, protein composition and microRNA content of human thymic exosomes. The thymic exosomes share characteristics with previously described exosomes such as antigen presentation molecules, but they also exhibit thymus specific features regarding surface markers, protein content and microRNA profile. Interestingly, thymic exosomes carry proteins that have a tissue restricted expression in the periphery which may suggest a role in T cell selection and the induction of central tolerance. We speculate that thymic exosomes may provide the means for intercellular information exchange necessary for negative selection and regulatory T cell formation of the developing thymocytes within the human thymic medulla.