Barbara Giovannone

CD8(+) T cells in the lesional skin of atopic dermatitis and psoriasis patients are an important source of IFN-γ, IL-13, IL-17, and IL-22.

Although CD4(+) T cells are known to contribute to the pathology of atopic dermatitis (AD) and psoriasis, the role of CD8(+) T cells in these diseases remains poorly characterized. The aim of this study was to characterize the cytokine production of T cells from AD and psoriasis skin. We found that CD4(+) T cells isolated from AD skin were largely Th2 (T helper type 2) biased, in agreement with prior reports. However, we also observed large numbers of CD8(+) T cells producing IL-13, IFN-γ, and IL-22. We observed increased numbers of CD8(+) T cells isolated from AD skin, and immunohistochemistry studies confirmed the presence of CD8(+) T cells in the dermis and epidermis of AD skin lesions. Surprisingly, T-cell cytokine production was similar in the lesional and nonlesional skin of patients with AD. T cells from psoriatic lesional skin predominantly produced IFN-γ, IL-17, and IL-22, in agreement with prior studies. However, in addition to Th17 cells, we observed high percentages of CD8(+) T cells that produced both IL-22 and IL-17 in psoriatic skin lesions. Our findings demonstrate that CD8(+) T cells are a significant and previously unappreciated source of inflammatory cytokine production in both AD and psoriasis.

A panel of biomarkers for disease severity in atopic dermatitis

A panel of biomarkers for disease severity in atopic dermatitis J. L. Thijs, S. Nierkens, A. Herath , C. A. F. Bruijnzeel-Koomen, E. F. Knol, B. Giovannone, M. S. de Bruin-Weller and D. Hijnen Department of Dermatology and Allergology, University Medical Center Utrecht, Utrecht, The Netherlands, U-DAIR and Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht, The Netherlands, MedImmune Biotech, Cambridge, UK and Department of Immunology, University Medical Center Utrecht, Utrecht, The Netherlands

Moving toward endotypes in atopic dermatitis: Identification of patient clusters based on serum biomarker analysis

Background Atopic dermatitis (AD) is a complex, chronic, inflammatory skin disease with a diverse clinical presentation. However, it is unclear whether this diversity exists at a biological level. Objective We sought to test the hypothesis that AD is heterogeneous at the biological level of individual inflammatory mediators. Methods Sera from 193 adult patients with moderate‐to‐severe AD (six area, six sign atopic dermatitis [SASSAD] score: geometric mean, 22.3 [95% CI, 21.3‐23.3] and 39.1 [95% CI, 37.5‐40.9], respectively) and 30 healthy control subjects without AD were analyzed for 147 serum mediators, total IgE levels, and 130 allergen‐specific IgE levels. Population heterogeneity was assessed by using principal component analysis, followed by unsupervised k‐means cluster analysis of the principal components. Results Patients with AD showed pronounced evidence of inflammation compared with healthy control subjects. Principal component analysis of data on sera from patients with AD revealed the presence of 4 potential clusters. Fifty‐seven principal components described approximately 90% of the variance. Unsupervised k‐means cluster analysis of the 57 largest principal components delivered 4 distinct clusters of patients with AD. Cluster 1 had high SASSAD scores and body surface areas with the highest levels of pulmonary and activation‐regulated chemokine, tissue inhibitor of metalloproteinases 1, and soluble CD14. Cluster 2 had low SASSAD scores with the lowest levels of IFN‐&agr;, tissue inhibitor of metalloproteinases 1, and vascular endothelial growth factor. Cluster 3 had high SASSAD scores with the lowest levels of IFN‐&bgr;, IL‐1, and epithelial cytokines. Cluster 4 had low SASSAD scores but the highest levels of the inflammatory markers IL‐1, IL‐4, IL‐13, and thymic stromal lymphopoietin. Conclusion AD is a heterogeneous disease both clinically and biologically. Four distinct clusters of patients with AD have been identified that could represent endotypes with unique biological mechanisms. Elucidation of these endotypes warrants further investigation and will require future intervention trials with specific agents, such as biologics.

TSLP is differentially regulated by vitamin D3 and cytokines in human skin

Thymic stromal lymphopoietin (TSLP) plays an important role in allergic diseases and is highly expressed in keratinocytes in human lesional atopic dermatitis (AD) skin. In nonlesional AD skin TSLP expression can be induced by applying house dust mite allergen onto the skin in the atopy patch test. Several studies have demonstrated that the induction of TSLP expression in mouse skin does not only lead to AD‐like inflammation of the skin, but also predisposes to severe inflammation of the airways. In mice, TSLP expression can be induced by application of the 1,25‐dihydroxyvitamin D3 (VD3) analogue calcipotriol and results in the development of eczema‐like lesions. The objective is to investigate the effect of VD3 (calcitriol) or calcipotriol on TSLP expression in normal human skin and skin from AD patients. Using multiple ex vivo experimental setups, the effects of calci(po)triol on TSLP expression by normal human skin, and skin from AD patients were investigated and compared to effects of calcipotriol on mouse and non‐human primates (NHP) skin. No induction of TSLP expression (mRNA or protein) was observed in human keratinocytes, normal human skin, nonlesional AD skin, or NHP skin samples after stimulation with calcipotriol or topical application of calcitriol. The biological activity of calci(po)triol in human skin samples was demonstrated by the increased expression of the VD3‐responsive Cyp24a1 gene. TSLP expression was induced by cytokines (IL‐4, IL‐13, and TNF‐α) in skin samples from all three species. In contrast to the findings in human and NHP, a consistent increase in TSLP expression was confirmed in mouse skin biopsies after stimulation with calcipotriol. VD3 failed to induce expression of TSLP in human or monkey skin in contrast to mouse, implicating careful extrapolation of this often‐used mouse model to AD patients.

Assessment of cyclobutane pyrimidine dimers by digital photography in human skin

UV-mediated DNA damage and repair are important mechanisms in research on UV-induced carcinogenesis. UV-induced DNA-damage and repair can be determined by immunohistochemical staining of photoproduct positive nuclei of keratinocytes in the epidermis. We developed a new method of analysing and quantifying thymine dimer (TT-CPD) positive cells in the epidermis. Normal skin of healthy controls was exposed to UVB ex vivo and in vivo. Skin samples were immunohistochemically stained for TT-CPDs. Digital images of the epidermis were quantified for TT-CPDs both visually and digitally. There was a UVB-dose dependent induction of TT-CPDs present in the ex vivo UVB-irradiated skin samples. The linear measurement range of the digital quantification was increased compared to the manual counting. The average 24-hour repair rate of the initiated TT-CPDs elicited by the UVB irradiation at T=0 of the 8 HCs showed a 34% decrease of TT-CPD photoproducts by the manual counting method and a 51% decrease determined by digital counting. The digital quantification method improves immunohistochemical quantification of DNA photo damage. It is more sensitive in measuring the extent of DNA-damage per nucleus.

Serum microRNA screening and functional studies reveal miR-483-5p as a potential driver of fibrosis in systemic sclerosis

OBJECTIVE MicroRNAs (miRNAs) are regulatory molecules, which have been addressed as potential biomarkers and therapeutic targets in rheumatic diseases. Here, we investigated the miRNA signature in the serum of systemic sclerosis (SSc) patients and we further assessed their expression in early stages of the disease. METHODS The levels of 758 miRNAs were evaluated in the serum of 26 SSc patients as compared to 9 healthy controls by using an Openarray platform. Three miRNAs were examined in an additional cohort of 107 SSc patients and 24 healthy donors by single qPCR. MiR-483-5p expression was further analysed in the serum of patients with localized scleroderma (LoS) (n = 22), systemic lupus erythematosus (SLE) (n = 33) and primary Sjögrens syndrome (pSS) (n = 23). The function of miR-483-5p was examined by transfecting miR-483-5p into primary human dermal fibroblasts and pulmonary endothelial cells. RESULTS 30 miRNAs were significantly increased in patients with SSc. Of these, miR-483-5p showed reproducibly higher levels in an independent SSc cohort and was also elevated in patients with preclinical-SSc symptoms (early SSc). Notably, miR-483-5p was not differentially expressed in patients with SLE or pSS, whereas it was up-regulated in LoS, indicating that this miRNA could be involved in the development of skin fibrosis. Consistently, miR-483-5p overexpression in fibroblasts and endothelial cells modulated the expression of fibrosis-related genes. CONCLUSIONS Our findings showed that miR-483-5p is up-regulated in the serum of SSc patients, from the early stages of the disease onwards, and indicated its potential function as a fine regulator of fibrosis in SSc.

Endoplasmic reticulum stress cooperates with Toll-like receptor ligation in driving activation of rheumatoid arthritis fibroblast-like synoviocytes

BackgroundEndoplasmic reticulum (ER) stress has proinflammatory properties, and transgenic animal studies of rheumatoid arthritis (RA) indicate its relevance in the process of joint destruction. Because currently available studies are focused primarily on myeloid cells, we assessed how ER stress might affect the inflammatory responses of stromal cells in RA.MethodsER stress was induced in RA fibroblast-like synoviocytes (FLS), dermal fibroblasts, and macrophages with thapsigargin or tunicamycin alone or in combination with Toll-like receptor (TLR) ligands, and gene expression and messenger RNA (mRNA) stability was measured by quantitative polymerase chain reaction. Cellular viability was measured using cell death enzyme-linked immunosorbent assays and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, and signaling pathway activation was analyzed by immunoblotting.ResultsNo cytotoxicity was observed in FLS exposed to thapsigargin, despite significant induction of ER stress markers. Screening of 84 proinflammatory genes revealed minor changes in their expression (fold change 90th percentile range 2.8–8.3) by thapsigargin alone, but the vast majority were hyperinduced during combined stimulation with thapsigargin and TLR ligands (35% greater than fivefold vs lipopolysaccharide alone). The synergistic response could not be explained by quantitative effects on nuclear factor-κB and mitogen-activated protein kinase pathways alone, but it was dependent on increased mRNA stability. mRNA stabilization was similarly enhanced by ER stress in dermal fibroblasts but not in macrophages, correlating with minimal cooperative effects on gene induction in macrophages.ConclusionsRA FLS are resistant to apoptosis induced by ER stress, but ER stress potentiates their activation by multiple TLR ligands. Interfering with downstream signaling pathway components of ER stress may be of therapeutic potential in the treatment of RA.

Effect of anticoagulants on 162 circulating immune related proteins in healthy subjects

HighlightsIn house validation shows inter‐assay variation <10%, and average inter‐assay variation of 12.2%Immune profiles remain stable after multiple freeze–thaw cycles.Only 19 out of 162 soluble proteins have similar expression in serum, EDTA plasma and sodium heparin plasma. &NA; Diagnosis of complex disease and response to treatment is often associated with multiple indicators, both clinical and laboratorial. With the use of biomarkers, various mechanisms have been unraveled which can lead to better and faster diagnosis, predicting and monitoring of response to treatment and new drug development. With the introduction of multiplex technology for immunoassays and the growing awareness of the role of immune‐monitoring during new therapeutic interventions it is now possible to test large numbers of soluble mediators in small sample volumes. However, standardization of sample collection and laboratory assessments remains suboptimal. We developed a multiplex immunoassay for detection of 162 immune related proteins in human serum and plasma. The assay was split in panels depending on natural occurring concentrations with a maximum of 60 proteins. The aim of this study was to evaluate precision, accuracy, reproducibility and stability of proteins when repeated freeze–thaw cycles are performed of this in‐house developed panel, as well as assessing the protein signature in plasma and serum using various anticoagulants. Intra‐assay variance of each mediator was <10%. Inter‐assay variance ranged between 1.6 and 37% with an average of 12.2%. Recoveries were similar for all mediators (mean 99.8 ± 2.6%) with a range between 89–107%. Next we measured all mediators in serum, EDTA plasma and sodium heparin plasma of 43 healthy control donors. Of these markers only 19 showed similar expression profiles in the 3 different matrixes. Only 5 mediators were effected by multiple freeze‐thawing cycles. Principal component analysis revealed different coagulants cluster separately and that sodium heparin shows the most consistent profile.

PD-1+CD8+ T cells are clonally expanding effectors in human chronic inflammation

Chronic inflammatory diseases are characterized by recurrent inflammatory attacks in the tissues mediated by autoreactive T cells. Identity and functional programming of CD8+ T cells at the target site of inflammation still remain elusive. One key question is whether, in these antigen-rich environments, chronic stimulation leads to CD8+ T cell exhaustion comparable to what is observed in infectious disease contexts. In the synovial fluid (SF) of juvenile idiopathic arthritis (JIA) patients, a model of chronic inflammation, an overrepresentation of PD-1+CD8+ T cells was found. Gene expression profiling, gene set enrichment analysis, functional studies, and extracellular flux analysis identified PD-1+CD8+ T cells as metabolically active effectors, with no sign of exhaustion. Furthermore, PD-1+CD8+ T cells were enriched for a tissue-resident memory (Trm) cell transcriptional profile and demonstrated increased clonal expansion compared with the PD-1– counterpart, suggesting antigen-driven expansion of locally adapted cells. Interestingly, this subset was also found increased in target tissues in other human chronic inflammatory diseases. These data indicate that local chronic inflammation drives the induction and expansion of CD8+ T cells endowed with potential detrimental properties. Together, these findings lay the basis for investigation of PD-1–expressing CD8+ T cell targeting strategies in human chronic inflammatory diseases.

Modulation of Lymphocyte Function In Vivo via Inhibition of Calcineurin or Purine Synthesis in Patients with Atopic Dermatitis

are indispensable for PG transporter activity, such as the missense mutations c.763G4A (p.Gly255Arg) and c.1668G4C (p.Gln556His) identified. Both changes are nonconservative, affect evolutionarily highly conserved amino acids (Figure 2), and were predicted in silico by all bioinformatic tools of pathogenic relevance (Supplementary Table 1 online). The pathogenic character of c.763G4A (p.Gly255Arg) and the impact of the glycine residue at position 255 is further strengthened by the fact that the same codon was mutated (c.764G4A; p.Gly255Glu) in a patient described by Zhang et al. (2012). To corroborate these data, we constructed molecular models of wild-type and mutated SLCO2A1 proteins to visualize changes of protein folding and structure (Meng et al., 2006; Bordoli et al., 2009; Yang et al., 2011). Both mutations are located in a transmembrane domain and considerably affect the protein structure (Figure 2), which was additionally confirmed by Ramachandran plots (Supplementary Figure S1 online). In this report, we show that SLCO2A1 is a frequent cause of autosomal recessive PHO, confirming the data recently published by Zhang et al., 2012. Although most patients harbor mutations in HPGD and SLCO2A1, further genetic heterogeneity is likely, and candidate approaches targeting other PG-signaling components seem promising. CONFLICT OF INTEREST JB, VF, NB, HB, and CB are employees of Bioscientia, a member of Sonic Healthcare.