RAQUEL LEAO ORFALI
Projetos de Pesquisa
Unidades Organizacionais
Instituto Central, Hospital das Clínicas, Faculdade de Medicina - Médico
LIM/56 - Laboratório de Investigação em Dermatologia e Imunodeficiências, Hospital das Clínicas, Faculdade de Medicina
LIM/56 - Laboratório de Investigação em Dermatologia e Imunodeficiências, Hospital das Clínicas, Faculdade de Medicina
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- Evidence of regulatory myeloid dendritic cells and circulating inflammatory epidermal dendritic cells-like modulated by Toll-like receptors 2 and 7/8 in adults with atopic dermatitis(2017) SANTOS, Vanessa G. dos; ORFALI, Raquel L.; TITZ, Tiago de Oliveira; DUARTE, Alberto J. da Silva; SATO, Maria N.; AOKI, ValeriaBackgroud Atopic dermatitis (AD) is a chronic, inflammatory skin disease characterized by intense pruritus and xerosis. Dendritic cells (DC) play an essential role in tissue inflammation in atopic dermatitis (AD) skin, especially the inflammatory epidermal dendritic cells (IDEC), a particular subset of myeloid dendritic cells (mDC). The aim of the present study was to assess the phenotype and function of mDC and circulating IDEC-like in peripheral blood mononuclear cells (PBMC) of adults with AD. Methods We selected 21 AD patients and 21 non-AD controls, age and gender matched. Expressions of Fc epsilon RI, CD36, TNF, IFN-gamma , and IL-10 in mDC were analyzed by flow cytometry under various stimuli, such as staphylococcal enterotoxin B (SEB), TLR2 (Pam3CSK4), TLR4 (LPS), and TLR7/8 (CL097) agonists. Results The most prominent findings in AD patients were: (i) enhanced frequency of IL-10 under TLR4 (LPS), and decreased frequency of IFN-gamma and TNF under TLR2 (Pam3CSK4) and 7/8 (CL097) stimulation in classic mDC; (ii) elevation of circulating IDEC-like frequency with TLR2 (Pam3CSK4) stimuli, augmented frequency of IFN-gamma in nonstimulated condition, and of IL-10 under TLR7/8 (CL097) stimuli in IDEC-like population. Conclusions In AD individuals, classic mDC showed an immunomodulatory profile, favoring tolerance in a combined action with IDEC-like, and inducing Th1 polarization. Our findings indicate a potential role of IDEC-like in the maintenance of inflammation in atopic dermatitis patients; moreover, IDEC-like may exert a regulatory impact on T cells of AD individuals through IL-10, often induced by agonist mimicking single stranded RNA virus.
- IgG from Adult Atopic Dermatitis (AD) Patients Induces Nonatopic Neonatal Thymic Gamma-Delta T Cells (gamma delta T) to Acquire IL-22/IL-17 Secretion Profile with Skin-Homing Properties and Epigenetic Implications Mediated by miRNA(2022) FAGUNDES, Beatriz Oliveira; SOUSA, Thamires Rodrigues de; NASCIMENTO, Andrezza; FERNANDES, Lorena Abreu; SGNOTTO, Fabio da Ressureicao; ORFALI, Raquel Leao; AOKI, Valeria; DUARTE, Alberto Jose da Silva; SANABANI, Sabri Saeed; VICTOR, Jefferson Russogamma delta T cells mature in the human thymus, and mainly produce IL-17A or IFN-gamma, but can also produce IL-22 and modulate a variety of immune responses. Here, we aimed to evaluate whether IgG from AD patients (AD IgG) can functionally modulate thymic nonatopic gamma delta T cells. Thymic tissues were obtained from 12 infants who had not had an atopic history. Thymocytes were cultured in mock condition, or in the presence of either AD IgG or therapeutic intravenous IgG (IVIg). Following these treatments, intracellular cytokine production, phenotype, and microRNA expression profiles were investigated. AD IgG could downregulate alpha 4 beta 7, upregulate CLA, and induce the production of IFN-gamma, IL-17, and IL-22 in gamma delta T cells. Although both AD IgG and IVIg could directly interact with gamma delta T cell membranes, AD IgG could reduce gamma delta T cell apoptosis. AD IgG could upregulate nine miRNAs compared to IVIg, and six when compared to the mock condition. In parallel, some miRNAs were downregulated. Target gene prediction and functional analysis indicated that some target genes were enriched in the negative regulation of cellular transcription. This study shows that AD IgG influences the production of IL-17 and IL-22 by intrathymic nonatopic gamma delta T cells, and demonstrates epigenetic implications mediated by miRNAs.
- Skin barrier in atopic dermatitis: beyond filaggrin(2016) ZANIBONI, Mariana Colombini; SAMORANO, Luciana Paula; ORFALI, Raquel Leao; AOKI, ValeriaAtopic dermatitis is a chronic inflammatory skin disease with a complex pathogenesis, where changes in skin barrier and imbalance of the immune system are relevant factors. The skin forms a mechanic and immune barrier, regulating water loss from the internal to the external environment, and protecting the individual from external aggressions, such as microorganisms, ultraviolet radiation and physical trauma. Main components of the skin barrier are located in the outer layers of the epidermis (such as filaggrin), the proteins that form the tight junction (TJ) and components of the innate immune system. Recent data involving skin barrier reveal new information regarding its structure and its role in the mechanic-immunological defense; atopic dermatitis (AD) is an example of a disease related to dysfunctions associated with this complex.
- Outlining the skin-homing and circulating CLA+NK cells in patients with severe atopic dermatitis(2024) LIMA, Josenilson Feitosa de; TEIXEIRA, Franciane Mouradian Emidio; RAMOS, Yasmim alefe Leuzzi; CARVALHO, Gabriel Costa de; BRANCO, Anna Claudia Calvielli Castelo; PEREIRA, Naiura Vieira; SOTTO, Mirian Nacagami; AOKI, Valeria; SATO, Maria Notomi; ORFALI, Raquel LeaoAtopic dermatitis (AD) is a complex, multifactorial skin disease, characterized by pruritus and predominant Th2 inflammation. Innate immune cells may play a role in AD development and are composed of granulocytes, macrophages, innate-like T cells, and innate lymphoid cells. This study investigates the phenotypic and functional profile of circulating CLA(+) natural killer (NK) cells and its role in the skin-homing to NK cells infiltrated in adults' skin with AD. We selected 44 AD patients and 27 non-AD volunteers for the study. The results showed increased frequencies of both CLA(+)CD56(bright) and CLA(+)CD56(dim) NK cell populations in the peripheral blood, mainly in severe AD patients. Upon SEB stimulation, we observed an augmented percentage of CLA(+)CD56(dim) NK cells expressing CD107a, IFN-gamma, IL-10, and TNF, reinforcing the role of staphylococcal enterotoxins in AD pathogenesis. Additionally, we demonstrated increased dermal expression of both NK cell markers NCAM-1/CD56 and pan-granzyme, corroborating the skin-homing, mostly in severe AD. Further studies are necessary to elucidate the potential role of NK cells in the chronification of the inflammatory process in AD skin, as well as their possible relationship with staphylococcal enterotoxins, and as practicable therapeutic targets.
- Increased expression of Filaggrin and Claudin-1 in the ocular surface of patients with atopic dermatitis(2022) CALLOU, T. M. P.; ORFALI, R. L.; SOTTO, M. N.; V, N. Pereira; ZANIBONI, M. C.; AOKI, V; BRITO, M. P.; MATSUDA, M.; SANTO, R. M.Background Atopic dermatitis (AD) is an itchy, chronic and inflammatory skin condition, with dysfunctional immune response and skin barrier defects. Reduction of filaggrin (FLG) and tight junctions (TJ) proteins, such as claudin-1 (CLDN-1), expression in cutaneous epithelial barrier is remarkable in AD pathogenesis. Ocular involvement occurs in approximately 40% of AD patients leading to changes in the structure of the conjunctiva. Objectives We aimed to evaluate the expression of FLG and CLDN-1 in the ocular surface of adults with AD, analysing bulbar conjunctival cells collected by a novel non-invasive cellular imprint. Methods Bulbar conjunctival epithelial cells were collected by cellular imprint technique, and FLG and CLDN-1 expression were assessed by immunofluorescence (IF) and real-time polymerase chain reaction (RT-PCR). Results We detected increased expression of FLG and CLDN-1, as well as their transcript levels in AD patients compared with healthy controls (HC). There was a positive correlation between tear film break-up time (TBUT) and FLG expression. Fluorescein staining was inversely associated with FLG expression. Conclusions Our results may reflect a reactive response of the ocular surface to AD-related ocular inflammation and associated dry eye disease. Further investigations focusing on the role of FLG and TJ expression in the ocular surface of AD patients may increment the understanding of the pathophysiology of extracutaneous AD and developing future targeted therapies.
- IgG from atopic dermatitis patients induces non-atopic infant thymic invariant natural killer T (iNKT) cells to produce IL-4, IL-17, and IL-10(2020) SANTOS, Ludimila S.; SGNOTTO, Fabio da Ressureicao; SOUSA, Thamires R.; ORFALI, Raquel L.; AOKI, Valeria; DUARTE, Alberto Jose da Silva; VICTOR, Jefferson R.Background Atopic dermatitis (AD) pathogenesis still needs to be elucidated, but invariant natural killer T (iNKT) cell involvement was already described by several groups. Our group has demonstrated that IgG antibodies purified from AD patients can modulate cytokine production by thymic T cells. Here we aimed to investigate if IgG from AD patients can modulate infant non-atopic thymic iNKT cells cytokine production in order to collaborate with the elucidation of AD development in infancy. Methods Thymic tissues were obtained from children from non-atopic mothers, and IgG was purified from AD patients diagnosed as moderate or severe and, as controls, from subjects clinically classified as non-atopic individuals. PBMCs from non-atopic individuals were also used in this study. Results Our results demonstrated that IgG from AD patients could induce non-atopic children thymic iNKT cells to produce higher levels of intracellular IL-4, IL-10, and IL-17 when compared to all control conditions. No effect was observed in non-atopic adults peripheral iNKT. We also observed that IgG from AD patients induces an increase in the expression of CD4 and Ror gamma t transcription factor in non-atopic children thymic iNKT cells compared to the condition of all controls. Conclusions These observations suggest that IgG from AD patients can induce a cytokine profile by thymic iNKT cells from non-atopic infants compatible with the observations in AD development, which can collaborate with the elucidation of AD pathogenesis.
conferenceObject A 25-year overview of the atopic dermatitis outpatient clinic at the University of Sao Paulo Medical School, Brazil(2014) ZANIBONI, M. C.; ORFALI, R. L.; TAKAOKA, R.; ISHIZAKI, A. S.; AOKI, V.conferenceObject IgG from atopic dermatitis patients induces IL-17 and IL-10 production in infant intra-thymic TCD4 and TCD8 cells(2018) SGNOTTO, F. R.; OLIVEIRA, M. G.; LIRA, A. A. L.; INOUE, A. H. S.; TITZ, T. O.; ORFALI, R. L.; BENTO-DE-SOUZA, L.; SATO, M. N.; AOKI, V.; DUARTE, A. J. S.; VICTOR, J. R.conferenceObject Atopic dermatitis in adults: Augmented circulating IgG4 and IgE antibodies against Staphylococcus aureus enterotoxin B(2013) ORFALI, R. L.; SATO, M. N.; SANTOS, V. G. Dos; TITZ, T. O.; DUARTE, A. S.; TAKAOKA, R.; AOKI, V.The aim of this study was to evaluate the profile of anti-Staphylococcus aureus enterotoxin B (SEB) antibody (Ab) response in adults with AD. We selected 38 patients diagnosed as AD (Hanifin & Rajka's criteria), aged between 18 and 65, and 26 healthy controls (HC), aged between 20 and 47 years. Severity of the disease was established according to EASI (Eczema Area and Severity Index) and patients were graded as mild (28%), moderate (58%) and severe (14%). Sera were assessed for IgG subclasses, IgA, IgM and IgE against SEB by ELISA. The humoral response in AD patients to SEB showed elevated circulating IgE and IgG4 levels (p < 0.05 and p δ 0.001, respectively), and decreased IgA and IgM levels (p < 0.05). Severity of atopic dermatitis was related with low IgG1 and IgG3 levels (p < 0.05), and high IgE antibody response (p < 0.05) to SEB. Interestingly, absence of IgE and IgG1 response to SEB was seen in some AD patients (26.3% and 18.4% respectively). In AD, the immunoglobulin-related subtype Th2 lymphocytes, such as IgE and IgG4, appear to be relevant in the response to superantigens. These results emphasize an altered pattern of Ab response to SEB in AD, which is impaired according the disease severity.- Staphylococcal enterotoxins modulate the effector CD4(+)T cell response by reshaping the gene expression profile in adults with atopic dermatitis(2019) ORFALI, Raquel Leao; YOSHIKAWA, Fabio Seiti Yamada; OLIVEIRA, Luanda Mara da Silva; PEREIRA, Natalli Zanete; LIMA, Josenilson Feitosa de; RAMOS, Yasmim Alefe Leuzzi; DUARTE, Alberto Jose da Silva; SATO, Maria Notomi; AOKI, ValeriaStaphylococcus aureus colonizes the skin of atopic dermatitis (AD) individuals, but the impact of its enterotoxins on the chronic activation of CD4(+)T cells demands further analysis. We aimed to analyze the CD4(+)T cell anergy profile and their phenotypic and functional features through differential expression of cellular activation markers, cytokine production and response to staphylococcal enterotoxin A (SEA). A panel of 84 genes relevant to T cell anergy was assessed by PCR array in FACS-sorted CD4(+)T cells, and the most prominent genes were validated by RT-qPCR. We evaluated frequencies of circulating CD4(+)T cells secreting single or multiple (polyfunctional) cytokines (IL-17A, IL-22, TNF, IFN-gamma, and MIP-1 beta) and expression of activation marker CD38 in response to SEA stimulation by flow cytometry. Our main findings indicated upregulation of anergy-related genes (EGR2 and IL13) promoted by SEA in AD patients, associated to a compromised polyfunctional response particularly in CD4(+)CD38(+)T cells in response to antigen stimulation. The pathogenic role of staphylococcal enterotoxins in adult AD can be explained by their ability to downmodulate the activated effector T cell response, altering gene expression profile such as EGR2 induction, and may contribute to negative regulation of polyfunctional CD4(+)T cells in these patients.