Single-cell RNA sequencing reveals 2D cytokine assay can model atopic dermatitis more accurately than immune-competent 3D setup.

Modelling atopic dermatitis (AD) in vitro is paramount to understand the disease pathophysiology and identify novel treatments. Previous studies have shown that the Th2 cytokines IL-4 and IL-13 induce AD-like features in keratinocytes in vitro. However, it has not been systematically researched whether the addition of Th2 cells, their supernatants or a 3D structure is superior to model AD compared to simple 2D cell culture with cytokines. For the first time, we investigated what in vitro option most closely resembles the disease in vivo based on single-cell RNA sequencing data (scRNA-seq) obtained from skin biopsies in a clinical study and published datasets of healthy and AD donors. In vitro models were generated with primary fibroblasts and keratinocytes, subjected to cytokine treatment or Th2 cell cocultures in 2D/3D. Gene expression changes were assessed using qPCR and Multiplex Immunoassays. Of all cytokines tested, incubation of keratinocytes and fibroblasts with IL-4 and IL-13 induced the closest in vivo-like AD phenotype which was observed in the scRNA-seq data. Addition of Th2 cells to fibroblasts failed to model AD due to the downregulation of ECM-associated genes such as POSTN. While keratinocytes cultured in 3D showed better stratification than in 2D, changes induced with AD triggers did not better resemble AD keratinocyte subtypes observed in vivo. Taken together, our comprehensive study shows that the simple model using IL-4 or IL-13 in 2D most accurately models AD in fibroblasts and keratinocytes in vitro, which may aid the discovery of novel treatment options.

Pituitary adenylate cyclase-activating polypeptide promotes cutaneous dendritic cell functions in contact hypersensitivity.

BACKGROUND: Sensory nerves regulate cutaneous local inflammation indirectly through induction of pruritus and directly by acting on local immune cells. The underlying mechanisms for how sensory nerves influence cutaneous acquired immune responses remain to be clarified. OBJECTIVE: This study aimed to explore the effect of peripheral nerves on cutaneous immune cells in cutaneous acquired immune responses. METHODS: We analyzed contact hypersensitivity (CHS) responses as a murine model of delayed-type hypersensitivity in absence or presence of resiniferatoxin-induced sensory nerve denervation. We conducted ear thickness measurements, flow cytometric analyses, and mRNA expression analyses in CHS. RESULTS: CHS responses were attenuated in mice that were denervated during the sensitization phase of CHS. By screening neuropeptides, we found that pituitary adenylate cyclase-activating polypeptide (PACAP) mRNA expression was decreased in the dorsal root ganglia after denervation. Administration of PACAP restored attenuated CHS response in resiniferatoxin-treated mice, and pharmacological inhibition of PACAP suppressed CHS. Flow cytometric analysis of skin-draining lymph nodes showed that cutaneous dendritic cell migration and maturation were reduced in both denervated mice and PACAP antagonist-treated mice. The expression of chemokine receptors CCR7 and CXCR4 of dendritic cell s was enhanced by addition of PACAP in vitro. CONCLUSION: These findings indicate that a neuropeptide PACAP promotes the development of CHS responses by inducing cutaneous dendritic cell functions during the sensitization phase.

The interplay between genetic and environmental factors in the pathogenesis of atopic dermatitis.

Atopic dermatitis (AD) is a chronic skin disorder characterized by pruritus and recurrent eczematous lesions that are accompanied by T-helper (Th)2-dominated inflammation. AD Etiology is not yet completely understood, but it is multifactorial. Moreover, the disease is characterized by complex interactions between genetic and environmental factors, such as skin barrier dysfunctions, allergy/immunity, and pruritus. For example, filaggrin is a key protein involved in skin barrier function. Th2 cells produce interleukin (IL)-31, which provokes pruritus, and other Th2 cytokines decrease filaggrin expression by keratinocytes. Dupilumab has recently been developed for AD treatment; its mechanism of action is to bind to IL-4 receptor α and inhibit downstream signaling induced by IL-4 and IL-13. This review summarizes the etiopathogenesis of AD and provides the rationale for selecting a novel targeted therapy.

IFN-α inhibits telomerase in human CD8⁺ T cells by both hTERT downregulation and induction of p38 MAPK signaling.

The cytokine IFN-α is secreted during viral infections and has been shown to inhibit telomerase activity and accelerate T cell differentiation in vivo. However, the mechanism for this inhibition is not clear. In this study, we show that IFN-α inhibits both the transcription and translation of human telomerase reverse transcriptase (hTERT), the catalytic component of telomerase, in activated CD8(+) T cells. This was associated with increased activity of the repressor of hTERT transcription E2 transcription factor and decreased activation of NF-κB that promotes hTERT transcription. However IFN-α did not affect the translocation of hTERT from the cytoplasm to the nucleus. IFN-α also inhibits AKT kinase activation but increases p38 MAPK activity, and both of these events have been shown previously to inhibit telomerase activity. Addition of BIRB796, an inhibitor of p38 activity, to IFN-α-treated cells reversed, in part, the inhibition of telomerase by this cytokine. Therefore, IFN-α can inhibit the enzyme telomerase in CD8(+) T cells by transcriptional and posttranslational mechanisms. Furthermore, the addition of IFN-α to CD8(+)CD27(+)CD28(+) T cells accelerates the loss of both these costimulatory molecules. This suggests that persistent viral infections may contribute to the accumulation of highly differentiated/senescent CD8(+)CD27(-)CD28(-) T cells during aging by promoting IFN-α secretion during repeated episodes of viral reactivation.

In vitro models for investigating itch.

Itch (pruritus) is a sensation that drives a desire to scratch, a behavior observed in many animals. Although generally short-lasting and not causing harm, there are several pathological conditions where chronic itch is a hallmark symptom and in which prolonged scratching can induce damage. Finding medications to counteract the sensation of chronic itch has proven difficult due to the molecular complexity that involves a multitude of triggers, receptors and signaling pathways between skin, immune and nerve cells. While much has been learned about pruritus from in vivo animal models, they have limitations that corroborate the necessity for a transition to more human disease-like models. Also, reducing animal use should be encouraged in research. However, conducting human in vivo experiments can also be ethically challenging. Thus, there is a clear need for surrogate models to be used in pre-clinical investigation of the mechanisms of itch. Most in vitro models used for itch research focus on the use of known pruritogens. For this, sensory neurons and different types of skin and/or immune cells are stimulated in 2D or 3D co-culture, and factors such as neurotransmitter or cytokine release can be measured. There are however limitations of such simplistic in vitro models. For example, not all naturally occurring cell types are present and there is also no connection to the itch-sensing organ, the central nervous system (CNS). Nevertheless, in vitro models offer a chance to investigate otherwise inaccessible specific cell-cell interactions and molecular pathways. In recent years, stem cell-based approaches and human primary cells have emerged as viable alternatives to standard cell lines or animal tissue. As in vitro models have increased in their complexity, further opportunities for more elaborated means of investigating itch have been developed. In this review, we introduce the latest concepts of itch and discuss the advantages and limitations of current in vitro models, which provide valuable contributions to pruritus research and might help to meet the unmet clinical need for more refined anti-pruritic substances.

Regulation of myeloperoxidase-specific T cell responses during disease remission in antineutrophil cytoplasmic antibody-associated vasculitis: the role of Treg cells and tryptophan degradation.

OBJECTIVE: T lymphocytes have been implicated in the pathogenesis of antineutrophil cytoplasmic antibody-associated vasculitis (AAV). Patients with myeloperoxidase (MPO) antineutrophil cytoplasmic antibody (ANCA) experience relapses less frequently than those with proteinase 3 ANCA, suggesting greater immune regulation. This study was undertaken to investigate MPO-specific T cell reactivity during disease remission and the factors regulating their responsiveness. METHODS: MPO-specific T cells were quantified by enzyme-linked immunospot assay with additional Treg cell depletion or exogenous interleukin-2. Serum tryptophan and its metabolites were measured. In vivo blockade of indoleamine 2,3-dioxygenase (IDO) was performed, and its effect on MPO reactivity was assessed. RESULTS: During disease remission, MPO-specific interferon-gamma-producing T cell frequencies were comparable with those found in healthy controls and significantly lower than those found in patients with acute disease. CD4+CD25+ regulatory cells did not play a role in maintaining these low MPO-specific T cell frequencies, since depletion of Treg cells did not augment MPO-specific responses, and FoxP3 levels were diminished in patients compared with controls. Treg cell function, however, was comparable in patients and controls, suggesting numerical rather than functional deficiency. We found diminished serum tryptophan levels and elevated levels of its metabolite kynurenine in patients with MPO AAV as compared with controls. To confirm the effect of tryptophan degradation on MPO responses in vivo, we inhibited degradation in MPO-immunized WKY rats and found greater immune responsiveness to MPO and a tendency to more severe glomerulonephritis. CONCLUSION: Our findings indicate that MPO-specific T cell frequencies are regulated during disease remission in association with tryptophan degradation. The tryptophan regulatory pathway is induced during active disease and persists during disease remission.

Anti-PD-1 and Anti-CTLA-4 Therapies in Cancer: Mechanisms of Action, Efficacy, and Limitations.

Melanoma, a skin cancer associated with high mortality rates, is highly radio- and chemotherapy resistant but can also be very immunogenic. These circumstances have led to a recent surge in research into therapies aiming to boost anti-tumor immune responses in cancer patients. Among these immunotherapies, neutralizing antibodies targeting the immune checkpoints T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) are being hailed as particularly successful. These antibodies have resulted in dramatic improvements in disease outcome and are now clinically approved in many countries. However, the majority of advanced stage melanoma patients do not respond or will relapse, and the hunt for the "magic bullet" to treat the disease continues. This review examines the mechanisms of action and the limitations of anti-PD-1/PD-L1 and anti-CTLA-4 antibodies which are the two types of checkpoint inhibitors currently available to patients and further explores the future avenues of their use in melanoma and other cancers.

The Phenomenology of the Diagnostic Process: A Primary Care-Based Survey.

BACKGROUND: While dichotomous tasks and related cognitive strategies have been extensively researched in cognitive psychology, little is known about how primary care practitioners (general practitioners [GPs]) approach ill-defined or polychotomous tasks and how valid or useful their strategies are. OBJECTIVE: To investigate cognitive strategies used by GPs for making a diagnosis. METHODS: In a cross-sectional study, we videotaped 282 consultations, irrespective of presenting complaint or final diagnosis. Reflective interviews were performed with GPs after each consultation. Recordings of consultations and GP interviews were transcribed verbatim and analyzed using a coding system that was based on published literature and systematically checked for reliability. RESULTS: In total, 134 consultations included 163 diagnostic episodes. Inductive foraging (i.e., the initial, patient-guided search) could be identified in 91% of consultations. It contributed an average 31% of cues obtained by the GP in 1 consultation. Triggered routines and descriptive questions occurred in 38% and 84% of consultations, respectively. GPs resorted to hypothesis testing, the hallmark of the hypothetico-deductive method, in only 39% of consultations. LIMITATIONS: Video recordings and interviews presumably interfered with GPs' behavior and accounts. GPs might have pursued more hypotheses and collected more information than usual. CONCLUSIONS: The testing of specific disease hypotheses seems to play a lesser role than previously thought. Our data from real consultations suggest that GPs organize their search for information in a skillfully adapted way. Inductive foraging, triggered routines, descriptive questions, and hypotheses testing are essential building blocks to make a diagnosis in the generalist setting.

Infiltration of PD-1-positive cells in combination with tumor site PD-L1 expression is a positive prognostic factor in cutaneous angiosarcoma.

Cutaneous angiosarcoma (CAS) is a malignant sarcoma with poor prognosis. Programmed cell death-1 (PD-1)/programmed cell death-1 ligand-1 (PD-L1) expression reflects antitumor immunity, and is associated with patient prognosis in various cancers. The purpose of this study is to investigate the relationship between PD-1/PD-L1 expression and CAS prognosis. CAS cases (n = 106) were immunohistochemically studied for PD-L1 and PD-1 expression, and the correlation with patient prognosis was analyzed. PD-L1 expression was assessed by flow cytometry on three CAS cell lines with or without IFNγ stimulation. A total of 30.2% of patients' samples were positive for PD-L1, and 17.9% showed a high infiltration of PD-1-positive cells. Univariate analysis showed a significant relationship between a high infiltration of PD-1-positive cells with tumor site PD-L1 expression and favorable survival in stage 1 patients (p = 0.014, log-rank test). Multivariable Cox-proportional hazard regression analysis also showed that patients with a high infiltration of PD-1-positive cells with tumor site PD-L1 expression were more likely to have favorable survival, after adjustment with possible confounders (hazard ratio (HR) = 0.38, p = 0.021, 95% confidence interval (CI) 0.16-0.86). Immunofluorescence staining of CAS samples revealed that PD-L1-positive cells were adjacent to PD-1-positive cells and/or tumor stroma with high IFNγ expression. In vitro stimulation with IFNγ increased PD-L1 expression in two out of three established CAS cell lines. Our results suggest that PD-1/PD-L1 expression is related to CAS progression, and the treatment with anti-PD-1 antibodies could be a new therapeutic option for CAS.

Peripheral blood Th9 cells are a possible pharmacodynamic biomarker of nivolumab treatment efficacy in metastatic melanoma patients.

Although nivolumab is associated with a significant improvement in overall survival and progression-free survival, only 20 to 40% of patients experience long-term benefit. It is therefore of great interest to identify a predictive marker of clinical benefit for nivolumab. To address this issue, the frequencies of CD4+ T cell subsets (Treg, Th1, Th2, Th9, Th17 and Th22), CD8+ T cells, and serum cytokine levels (IFNγ, IL-4, IL-9, IL-10, TGF-ß) were assessed in 46 patients with melanoma. Eighteen patients responded to nivolumab, and the other 28 patients did not. An early increase in Th9 cell counts during the treatment with nivolumab was associated with an improved clinical response. Before the first nivolumab infusion, the responders displayed elevated serum concentrations of TGF-ß compared to non-responders. Th9 induction by IL-4 and TGF-ß was enhanced by PD-1/PD-L1 blockade in vitro. The role of IL-9 in disease progression was further assessed using a murine melanoma model. In vivo IL-9 blockade promoted melanoma progression in mice using an autochthonous mouse melanoma model, and the cytotoxic ability of murine melanoma-specific CD8+ T cells was enhanced in the presence of IL-9 in vitro. These findings suggest that Th9 cells, which produce IL-9, play an important role in the successful treatment of melanoma patients with nivolumab. Th9 cells therefore represent a valid biomarker to be further developed in the setting of anti-PD-1 therapy.

The Characterization of Varicella Zoster Virus-Specific T Cells in Skin and Blood during Aging.

Reactivation of the varicella zoster virus (VZV) increases during aging. Although the effects of VZV reactivation are observed in the skin (shingles), the number and functional capacity of cutaneous VZV-specific T cells have not been investigated. The numbers of circulating IFN-γ-secreting VZV-specific CD4(+) T cells are significantly decreased in old subjects. However, other measures of VZV-specific CD4(+) T cells, including proliferative capacity to VZV antigen stimulation and identification of VZV-specific CD4(+) T cells with an major histocompatibility complex class II tetramer (epitope of IE-63 protein), were similar in both age groups. The majority of T cells in the skin of both age groups expressed CD69, a characteristic of skin-resident T cells. VZV-specific CD4(+) T cells were significantly increased in the skin compared with the blood in young and old subjects, and their function was similar in both age groups. In contrast, the number of Foxp3(+) regulatory T cells and expression of the inhibitory receptor programmed cell death -1 PD-1 on CD4(+) T cells were significantly increased in the skin of older humans. Therefore, VZV-specific CD4(+) T cells in the skin of older individuals are functionally competent. However, their activity may be restricted by multiple inhibitory influences in situ.

Expression profiling the temperature-dependent amphibian response to infection by Batrachochytrium dendrobatidis.

Amphibians are experiencing a panzootic of unprecedented proportions caused by the emergence of Batrachochytrium dendrobatidis (Bd). However, all species are not equally at risk of infection, and risk is further modified by environmental variables, specifically temperature. In order to understand how, and when, hosts mount a response to Bd we analysed infection dynamics and patterns of gene expression in the model amphibian species Silurana (Xenopus) tropicalis. Mathematical modelling of infection dynamics demonstrate the existence of a temperature-dependent protective response that is largely independent of the intrinsic growth-rate of Bd. Using temporal expression-profiling by microarrays and qRT-PCR, we characterise this response in the main amphibian lymphoid tissue, the spleen. We demonstrate that clearance of Bd at the host-optimal temperature is not clearly associated with an adaptive immune response, but rather is correlated with the induction of components of host innate immunity including the expression of genes that are associated with the production of the antimicrobial skin peptide preprocareulein (PPCP) as well as inflammatory responses. We find that adaptive immunity appears to be lacking at host-optimal temperatures. This suggests that either Bd does not stimulate, or suppresses, adaptive immunity, or that trade-offs exist between innate and adaptive limbs of the amphibian immune system. At cold temperatures, S. tropicalis loses the ability to mount a PPCP-based innate response, and instead manifests a more pronounced inflammatory reaction that is characterised by the production of proteases and higher pathogen burdens. This study demonstrates the temperature-dependency of the amphibian response to infection by Bd and indicates the influence that changing climates may exert on the ectothermic host response to pathogens.