An overview of S100 proteins and their functions in skin homeostasis, interface dermatitis conditions and other skin pathologies.

S100 proteins comprise a family of structurally related proteins that are calcium-sensitive. S100 proteins have been found to play various roles in regulation of cell apoptosis, cell proliferation and differentiation, cell migration and invasion, energy metabolism, calcium homeostasis, protein phosphorylation, anti-microbial activity and inflammation in a variety of cell types. While the specific function of many S100 proteins remains unknown, some of the S100 proteins serve as disease biomarkers as well as possible therapeutic targets in skin diseases. Interface dermatitis (ID) is a histopathological term that covers many different skin conditions including cutaneous lupus erythematosus, lichen planus, and dermatomyositis. These pathologies share similar histological features, which include basal cell vacuolization and lymphocytic infiltration at the dermal-epidermal junction. In this review, we summarize how the S100 protein family contributes to both homeostatic and inflammatory processes in the skin. We also highlight the role of S100 proteins in neuronal signalling, describing how this might contribute to neuroimmune interactions in ID and other skin pathologies. Last, we discuss what is known about the S100 family proteins as both biomarkers and potential treatment targets in specific pathologies.

T-cell positioning by chemokines in autoimmune skin diseases.

Autoimmune skin diseases are complex processes in which autoreactive cells must navigate through the skin tissue to find their targets. Regulatory T cells in the skin help to mitigate autoimmune inflammation and may in fact be responsible for the patchy nature of these conditions. In this review, we will discuss chemokines that are important for global recruitment of T cell populations to the skin during disease, as well as signals that fine-tune their localization and function. We will describe prototypical disease responses and chemokine families that mediate these responses. Lastly, we will include an overview of chemokine-targeting drugs that have been tested as new treatment strategies for autoimmune skin diseases.

Suction blistering the lesional skin of vitiligo patients reveals useful biomarkers of disease activity.

BACKGROUND: Vitiligo is an autoimmune disease of the skin with limited treatment options; there is an urgent need to identify and validate biomarkers of disease activity to support vitiligo clinical studies. OBJECTIVE: To investigate potential biomarkers of disease activity directly in the skin of vitiligo subjects and healthy subjects. METHODS: Patient skin was sampled via a modified suction-blister technique, allowing for minimally invasive, objective assessment of cytokines and T-cell infiltrates in the interstitial skin fluid. Potential biomarkers were first defined and later validated in separate study groups. RESULTS: In screening and validation, CD8+ T-cell number and C-X-C motif chemokine ligand (CXCL) 9 protein concentration were significantly elevated in active lesional compared to nonlesional skin. CXCL9 protein concentration achieved greater sensitivity and specificity by receiver operating characteristic analysis. Suction blistering also allowed for phenotyping of the T-cell infiltrate, which overwhelmingly expresses C-X-C motif chemokine receptor 3. LIMITATIONS: A small number of patients were enrolled for the study, and only a single patient was used to define the treatment response. CONCLUSION: Measuring CXCL9 directly in the skin might be effective in clinical trials as an early marker of treatment response. Additionally, use of the modified suction-blister technique supports investigation of inflammatory skin diseases using powerful tools like flow cytometry and protein quantification.

Mannose-capped lipoarabinomannan from Mycobacterium tuberculosis preferentially inhibits sphingosine-1-phosphate-induced migration of Th1 cells.

Chemokine receptor cross-desensitization provides an important mechanism to regulate immune cell recruitment at sites of inflammation. We previously reported that the mycobacterial cell wall glycophospholipid mannose-capped lipoarabinomannan (ManLAM) could induce human peripheral blood T cell chemotaxis. Therefore, we examined the ability of ManLAM to desensitize T cells to other chemoattractants as a potential mechanism for impaired T cell homing and delayed lung recruitment during mycobacterial infection. We found that ManLAM pretreatment inhibited in vitro migration of naive human or mouse T cells to the lymph node egress signal sphingosine-1-phosphate (S1P). Intratracheal administration of ManLAM in mice resulted in significant increases in T cells, primarily CCR5(+) (Th1) cells, in lung-draining lymph nodes. To investigate the selective CCR5 effect, mouse T cells were differentiated into Th1 or Th2 populations in vitro, and their ability to migrate to S1P with or without ManLAM pretreatment was analyzed. ManLAM pretreatment of Th1 populations inhibited S1P-induced migration but had no effect on Th2 cell S1P-directed migration, suggesting a differential effect by S1P on the two subsets. The PI3K/AKT inhibitor Ly294002 inhibited S1P-directed migration by Th1 cells, whereas the ERK inhibitor U0126 inhibited Th2 cell S1P-directed migration. These observations demonstrate that S1P-induced migratory responses in Th1 and Th2 lymphocytes occurs via different signaling pathways and suggests further that the production of ManLAM during Mycobacterium tuberculosis infection may function to sequester Th1 cells in lung-draining lymph nodes, thereby delaying their recruitment to the lung.

Targeted proteomics of cerebrospinal fluid in treatment naïve multiple sclerosis patients identifies immune biomarkers of clinical phenotypes.

Multiple sclerosis (MS) is an inflammatory demyelinating disease with heterogeneous clinical presentations and variable long-term disability accumulation. There are currently no standard criteria to accurately predict disease outcomes. In this study we investigated the cross-sectional relationship between disease phenotype and immune-modulating cytokines and chemokines in cerebrospinal fluid (CSF). We analyzed CSF from 20 DMT-naïve MS patients using Olink Proteomics' Target 96 Inflammation panel and correlated the resulting analytes with respect to (1) disease subtype, (2) patient age and sex, (3) extent of clinical disability, and (4) MRI segmental brain volumes. We found that intrathecal IL-4 correlated with higher Expanded Disability Status Scale (EDSS) scores and longer 25-foot walk times, and CD8A correlated with decreased thalamic volumes and longer 9-hole peg test times. Male sex was associated with higher FGF-19 expression, and Tumefactive MS with elevated CCL4. Several inflammatory markers were correlated with older age at the time of LP. Finally, higher intrathecal IL-33 correlated with increased MS lesion burden and multi-compartment brain atrophy. This study confirms immune heterogeneity underlying CSF profiles in MS, but also identifies several inflammatory protein biomarkers that may be of use for predicting clinical outcomes in future algorithms.

Baseline tumor gene expression signatures correlate with chemoimmunotherapy treatment responsiveness in canine B cell lymphoma.

Pet dogs develop spontaneous diffuse large B cell lymphoma (DLBCL), and veterinary clinical trials have been employed to treat canine DLBCL and to inform clinical trials for their human companions. A challenge that remains is selection of treatment to improve outcomes. The dogs in this study were part of a larger clinical trial evaluating the use of combinations of doxorubicin chemotherapy, anti-CD20 monoclonal antibody, and one of three small molecule inhibitors: KPT-9274, TAK-981, or RV1001. We hypothesized that significant differential expression of genes (DEGs) in the tumors at baseline could help predict which dogs would respond better to each treatment based on the molecular pathways targeted by each drug. To this end, we evaluated gene expression in lymph node aspirates from 18 trial dogs using the NanoString nCounter Canine Immuno-oncology (IO) Panel. We defined good responders as those who relapsed after 90 days, and poor responders as those who relapsed prior to 90 days. We analyzed all dogs at baseline and compared poor responders to good responders, and found increased CCND3 correlated with poor prognosis and increased CD36 correlated with good prognosis, as is observed in humans. There was minimal DEG overlap between treatment arms, prompting separate analyses for each treatment cohort. Increased CREBBP and CDKN1A for KPT-9274, increased TLR3 for TAK-981, and increased PI3Kδ, AKT3, and PTEN, and decreased NRAS for RV1001 were associated with better prognoses. Trends for selected candidate biomarker genes were confirmed via qPCR. Our findings emphasize the heterogeneity in DLBCL, similarities and differences between canine and human DLBCL, and ultimately identify biomarkers that may help guide the choice of chemoimmunotherapy treatment in dogs.

Cathepsin W, T-cell receptor-associated transmembrane adapter 1, lymphotactin and killer cell lectin like receptor K1 are sensitive and specific RNA biomarkers of canine epitheliotropic lymphoma.

Cutaneous T-cell lymphoma (CTCL) is an uncommon type of lymphoma involving malignant skin-resident or skin-homing T cells. Canine epitheliotropic lymphoma (EL) is the most common form of CTCL in dogs, and it also spontaneously arises from T lymphocytes in the mucosa and skin. Clinically, it can be difficult to distinguish early-stage CTCLs apart from other forms of benign interface dermatitis (ID) in both dogs and people. Our objective was to identify novel biomarkers that can distinguish EL from other forms of ID, and perform comparative transcriptomics of human CTCL and canine EL. Here, we present a retrospective gene expression study that employed archival tissue from biorepositories. We analyzed a discovery cohort of 6 canines and a validation cohort of 8 canines with EL which occurred spontaneously in client-owned companion dogs. We performed comparative targeted transcriptomics studies using NanoString to assess 160 genes from lesional skin biopsies from the discovery cohort and 800 genes from the validation cohort to identify any significant differences that may reflect oncogenesis and immunopathogenesis. We further sought to determine if gene expression in EL and CTCL are conserved across humans and canines by comparing our data to previously published human datasets. Similar chemokine profiles were observed in dog EL and human CTCL, and analyses were performed to validate potential biomarkers and drivers of disease. In dogs, we found enrichment of T cell gene signatures, with upregulation of IFNG, TNF, PRF1, IL15, CD244, CXCL10, and CCL5 in EL in dogs compared to healthy controls. Importantly, CTSW, TRAT1 and KLRK1 distinguished EL from all other forms of interface dermatitis we studied, providing much-needed biomarkers for the veterinary field. XCL1/XCL2 were also highly specific of EL in our validation cohort. Future studies exploring the oncogenesis of spontaneous lymphomas in companion animals will expand our understanding of these disorders. Biomarkers may be useful for predicting disease prognosis and treatment responses. We plan to use our data to inform future development of targeted therapies, as well as for repurposing drugs for both veterinary and human medicine.

CXCL9 Links Skin Inflammation and Fibrosis through CXCR3-Dependent Upregulation of Col1a1 in Fibroblasts.

Morphea is characterized by initial inflammation followed by fibrosis of the skin and soft tissue. Despite its substantial morbidity, the pathogenesis of morphea is poorly studied. Previous work showed that CXCR3 ligands CXCL9 and CXCL10 are highly upregulated in the sera and lesional skin of patients with morphea. We found that an early inflammatory subcutaneous bleomycin mouse model of dermal fibrosis mirrors the clinical, histological, and immune dysregulation observed in human morphea. We used this model to examine the role of the CXCR3 chemokine axis in the pathogenesis of cutaneous fibrosis. Using the REX3 (Reporting the Expression of CXCR3 ligands) mice, we characterized which cells produce CXCR3 ligands over time. We found that fibroblasts contribute the bulk of CXCL9-RFP and CXCL10-BFP by percentage, whereas macrophages produce high amounts on a per-cell basis. To determine whether these chemokines are mechanistically involved in pathogenesis, we treated Cxcl9-, Cxcl10-, or Cxcr3-deficient mice with bleomycin and found that fibrosis is dependent on CXCL9 and CXCR3. Addition of recombinant CXCL9 but not CXCL10 to cultured mouse fibroblasts induced Col1a1 mRNA expression, indicating that the chemokine itself contributes to fibrosis. Taken together, our studies provide evidence that CXCL9 and its receptor CXCR3 are functionally required for inflammatory fibrosis.

Rational design of a JAK1-selective siRNA inhibitor for the modulation of autoimmunity in the skin.

Inhibition of Janus kinase (JAK) family enzymes is a popular strategy for treating inflammatory and autoimmune skin diseases. In the clinic, small molecule JAK inhibitors show distinct efficacy and safety profiles, likely reflecting variable selectivity for JAK subtypes. Absolute JAK subtype selectivity has not yet been achieved. Here, we rationally design small interfering RNAs (siRNAs) that offer sequence-specific gene silencing of JAK1, narrowing the spectrum of action on JAK-dependent cytokine signaling to maintain efficacy and improve safety. Our fully chemically modified siRNA supports efficient silencing of JAK1 expression in human skin explant and modulation of JAK1-dependent inflammatory signaling. A single injection into mouse skin enables five weeks of duration of effect. In a mouse model of vitiligo, local administration of the JAK1 siRNA significantly reduces skin infiltration of autoreactive CD8+ T cells and prevents epidermal depigmentation. This work establishes a path toward siRNA treatments as a new class of therapeutic modality for inflammatory and autoimmune skin diseases.

Mannose-capped Lipoarabinomannan from Mycobacterium tuberculosis induces soluble tumor necrosis factor receptor production through tumor necrosis factor alpha-converting enzyme activation.

Primary Mycobacterium tuberculosis infection results in granuloma formation in lung tissue. A granuloma encapsulates mycobacterium-containing cells, thereby preventing dissemination and further infection. Tumor necrosis factor alpha (TNF-α) is a host-protective cytokine during M. tuberculosis infection due to its role in promoting and sustaining granuloma formation. TNF activity is regulated through the production of soluble TNF receptors (sTNFRI and sTNFRII). Therefore, we examined the potential production of endogenous sTNFRs during M. tuberculosis infection. Using the murine model of aerosol M. tuberculosis infection, we determined that levels of sTNFR production were elevated in bronchoalveolar lavage fluid 1 month following infection. An investigation of M. tuberculosis cell wall components identified that the known virulence factor mannose-capped lipoarabinomannan (ManLAM) was sufficient to induce sTNFR production, with sTNFRII being produced preferentially compared with sTNFRI. ManLAM stimulated the release of sTNFRs without TNF production, which corresponded to an increase in TNF-α-converting enzyme (TACE) activity. To determine the relevance of these findings, serum samples from M. tuberculosis-infected patients were tested and found to have an increase in the sTNFRII/sTNFRI ratio. These data identify a mechanism by which M. tuberculosis infection can promote the neutralization of TNF and furthermore suggest the potential use of the sTNFRII/sTNFRI ratio as an indicator of tuberculosis disease.

Evaluating the use of JAK inhibitors in inflammatory connective tissue diseases in pediatric patients: an update.

INTRODUCTION: Connective tissue diseases (CTDs) are a category of conditions that affect tissues that support and provide structure to the body. These diseases include rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, and sclerosing diseases. CTDs can be caused by dysregulation of inflammatory pathways, specifically an upregulation of interferons and JAK/STAT pathway activation. AREAS COVERED: While CTDs have historically been treated with broadly immunosuppressant medications such as corticosteroids and disease-modifying antirheumatic drugs (DMARDS), newer and more targeted immunomodulating medications called Janus kinase inhibitors (JAKi) have emerged as potential treatments. EXPERT OPINION: While most studies regarding JAKi for CTDs have focused on adult populations, pediatric patients with CTDs may also benefit from JAKi therapy. Moreover, the JAK/STAT inhibitor tofacitinib has been approved by the FDA for the treatment of active polyarticular course juvenile idiopathic arthritis. In this review, we have summarized what has been published on the use of JAKi for various pediatric CTDs.

Medicinal Plant Extracts and Natural Compounds for the Treatment of Cutaneous Lupus Erythematosus: A Systematic Review.

Cutaneous lupus erythematosus (CLE) is a group of autoimmune connective tissue disorders that significantly impact quality of life. Current treatment approaches typically use antimalarial medications, though patients may become recalcitrant. Other treatment options include general immunosuppressants, highlighting the need for more and more targeted treatment options. The purpose of this systematic review was to identify potential compounds that could be repurposed for CLE from natural products since many rheumatologic drugs are derived from natural products, including antimalarials. This study was registered with PROSPERO, the international prospective register of systematic reviews (registration number CRD42021251048). We comprehensively searched Ovid Medline, Cochrane Library, and Scopus databases from inception to April 27th, 2021. These terms included cutaneous lupus erythematosus; general plant, fungus, bacteria terminology; selected plants and plant-derived products; selected antimalarials; and JAK inhibitors. Our search yielded 13,970 studies, of which 1,362 were duplicates. We screened 12,608 abstracts, found 12,043 to be irrelevant, and assessed 565 full-text studies for eligibility. Of these, 506 were excluded, and 59 studies were included in the data extraction. The ROBINS-I risk of bias assessment tool was used to assess studies that met our inclusion criteria. According to our findings, several natural compounds do reduce inflammation in lupus and other autoimmune skin diseases in studies using in vitro methods, mouse models, and clinical observational studies, along with a few randomized clinical trials. Our study has cataloged evidence in support of potential natural compounds and plant extracts that could serve as novel sources of active ingredients for the treatment of CLE. It is imperative that further studies in mice and humans are conducted to validate these findings. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=251048.

A Keratinocyte-Tethered Biologic Enables Location-Precise Treatment in Mouse Vitiligo.

Despite the central role of IFN-γ in vitiligo pathogenesis, systemic IFN-γ neutralization is an impractical treatment option owing to strong immunosuppression. However, most patients with vitiligo present with <20% affected body surface area, which provides an opportunity for localized treatments that avoid systemic side effects. After identifying keratinocytes as key cells that amplify IFN-γ signaling during vitiligo, we hypothesized that tethering an IFN-γ‒neutralizing antibody to keratinocytes would limit anti‒IFN-γ effects on the treated skin for the localized treatment. To that end, we developed a bispecific antibody capable of blocking IFN-γ signaling while binding to desmoglein expressed by keratinocytes. We characterized the effect of the bispecific antibody in vitro, ex vivo, and in a mouse model of vitiligo. Single-photon emission computed tomography/computed tomography biodistribution and serum assays after local footpad injection revealed that the bispecific antibody had improved skin retention, faster elimination from the blood, and less systemic IFN-γ inhibition than the nontethered version. Furthermore, the bispecific antibody conferred localized protection almost exclusively to the treated footpad during vitiligo, which was not possible by local injection of the nontethered anti‒IFN-γ antibody. Thus, keratinocyte tethering proved effective while significantly diminishing the off-tissue effects of IFN-γ blockade, offering a safer treatment strategy for localized skin diseases, including vitiligo.

Th2 to Th1 Transition Is Required for Induction of Skin Lesions in an Inducible and Recurrent Murine Model of Cutaneous Lupus-Like Inflammation.

Cutaneous lupus erythematosus (CLE) is an autoimmune skin disease characterized by a strong IFN signature, normally associated with type I IFNs. However, increasing evidence points to an additional role for IFNγ, or at least a pathogenic T effector subset dependent on IFNγ, for disease progression. Nevertheless, Th2 effector subsets have also been implicated in CLE. We have now assessed the role of specific T cell subsets in the initiation and persistence of skin disease using a T cell-inducible murine model of CLE, dependent on KJ1-26 T cell recognition of an ovalbumin fusion protein. We found that only Th2-skewed cells, and not Th1-skewed cells, induced the development of skin lesions. However, we provide strong evidence that the Th2 disease-initiating cells convert to a more Th1-like functional phenotype in vivo by the time the skin lesions are apparent. This phenotype is maintained and potentiates over time, as T cells isolated from the skin, following a second induction of self-antigen, expressed more IFN-γ than T cells isolated at the time of the initial response. Transcriptional analysis identified additional changes in the KJ1-26 T cells at four weeks post injection, with higher expression levels of interferon stimulated genes (ISGs) including CXCL9, IRF5, IFIH1, and MX1. Further, injection of IFN-γ-/- T cells faied to induce skin disease in mice. We concluded that Th2 cells trigger skin lesion formation in CLE, and these cells switch to a Th1-like phenotype in the context of a TLR7-driven immune environment that is stable within the T cell memory compartment.

Using Gene Expression Analysis to Understand Complex Autoimmune Skin Disease Patients: A Series of Four Canine Cutaneous Lupus Erythematosus Cases.

Cutaneous Lupus Erythematosus (CLE) is an autoimmune skin disease that occurs in almost two-thirds of people with Systemic Lupus Erythematosus (SLE) and can exist as its own entity. Despite its negative impact on the quality of life of patients, lupus pathogenesis is not fully understood. In recent years, the role of gene expression analysis has become important in understanding cellular functions and disease causation within and across species. Interestingly, dogs also develop CLE, providing a spontaneous animal model of disease. Here, we present a targeted transcriptomic analysis of skin biopsies from a case series of four dogs with complex autoimmunity with suspected CLE. We identified 92 differentially expressed genes (DEGs), including type 1 interferon, B cell, and T cell-related genes, in the four cases compared to healthy skin margin controls. Additionally, we compared our results with existing CLE datasets from humans and mice and found that humans and canines share 49 DEGs, whereas humans and mice shared only 25 DEGs in our gene set. Immunohistochemistry of IFNG and CXCL10, two of the most highly upregulated inflammatory mediators, confirmed protein-level expression and revealed immune cells as the primary source of CXCL10 in dogs with SLE, whereas keratinocytes stained strongly for CXCL10 in dogs without SLE. We propose that gene expression analysis may aid the diagnosis of complex autoimmune skin diseases and that dogs may provide important insights into CLE and SLE pathogeneses, or more broadly, skin manifestations during systemic autoimmunity.

Immunopathogenesis of alopecia areata.

Alopecia areata (AA) is an autoimmune disorder resulting in hair loss. It has numerous variants or patterns, including diffuse type, patchy type, AA totalis, AA universalis, and more. In this graphical review, we provide an overview of AA immunopathogenesis, highlighting loss of immune privilege in the hair follicle as well as key immune cell types, cytokines and chemokines that drive autoimmune attack of the hair follicle. We also summarize recent literature identifying agents that block these pathways that could serve as new immunomodulatory treatments for AA.

Resident Memory T Cells in Autoimmune Skin Diseases.

Tissue resident memory T cells (TRM) are a critical component of the immune system, providing the body with an immediate and highly specific response against pathogens re-infecting peripheral tissues. More recently, however, it has been demonstrated that TRM cells also form during autoimmunity. TRM mediated autoimmune diseases are particularly destructive, because unlike foreign antigens, the self-antigens are never cleared, continuously activating self-reactive TRM T cells. In this article, we will focus on how TRMs mediate disease in autoimmune skin conditions, specifically vitiligo, psoriasis, cutaneous lupus erythematosus, alopecia areata and frontal fibrosing alopecia.

Type I interferon signaling limits viral vector priming of CD8+ T cells during initiation of vitiligo and melanoma immunotherapy.

Vitiligo is an autoimmune skin disease in which epidermal melanocytes are targeted for destruction by CD8+ T cells specific for melanocyte/melanoma-shared antigens. IFNγ is the central cytokine driving disease, but the role of type I IFN in vitiligo remains unclear. We investigated the functional role of type I IFN during vitiligo progression using two different mouse models: one induced with a vaccinia virus (VV) vaccine and one induced with dendritic cells to prime autoimmune T cells. Induction of vitiligo by VV in IFNaR-deficient mice led to the development of severe vitiligo compared with wild-type (WT) mice and was characterized by a significantly enhanced effector CD8+ T-cell response. Severe vitiligo in this model was a result of VV persistence, because exacerbation of disease in IFNaR-deficient mice was not observed when antigen-pulsed dendritic cells were used to induce vitiligo instead of virus. Treatment of B16F10 melanoma-inoculated mice with VV vaccine therapy also induced a significantly enhanced anti-tumor response in IFNaR-deficient mice compared with WT. These results not only help define the pathways responsible for vitiligo progression but also suggest that blockade of type I IFNs following administration of a VV vaccine may provide increased immunogenicity and efficacy for melanoma immunotherapy.

Upcoming treatments for morphea.

Morphea (localized scleroderma) is a rare autoimmune connective tissue disease with variable clinical presentations, with an annual incidence of 0.4-2.7 cases per 100,000. Morphea occurs most frequently in children aged 2-14 years, and the disease exhibits a female predominance. Insights into morphea pathogenesis are often extrapolated from studies of systemic sclerosis due to their similar skin histopathologic features; however, clinically they are two distinct diseases as evidenced by different demographics, clinical features, disease course and prognosis. An interplay between genetic factors, epigenetic modifications, immune and vascular dysfunction, along with environmental hits are considered as the main contributors to morphea pathogenesis. In this review, we describe potential new therapies for morphea based on both preclinical evidence and ongoing clinical trials. We focus on different classes of therapeutics, including antifibrotic, anti-inflammatory, cellular and gene therapy, and antisenolytic approaches, and how these target different aspects of disease pathogenesis.