Contribution to the study of inflammasome and programmed cell death in paracoccidioidomycosis oral lesions.

BACKGROUND: Paracoccidioidomycosis is the most prevalent systemic mycosis in Latin America, with a high incidence in Brazil, Colombia and Venezuela, and constitutes a serious public health problem, a frequent cause of morbidity and disability for work. Some mechanisms of cell death are described as important tools in infectious processes. When apoptosis is blocked, RIPK (Receptor-interacting protein kinase) 3 dependent, a caspase-independent form of cell death, can limit the replication and spread of pathogens. Some molecules that mediate necroptosis include RIPK3 and have been extensively studied due to their signalling mechanism and pathological function. RIPK3 activates NLRP1 and NLRP3-mediated inflammasome formation. Caspase-1 has an important role in processing the cytokines ILß and IL18 to their active form. Such molecules are part of the inflammasome characterization, whose caspase-1-dependent activation promotes the death of pyroptotic cells and the secretion of proinflammatory cytokines. Knowledge about the mechanisms of pathogen-mediated cell death can be useful for understanding of the pathogenesis of infections and inflammatory conditions. OBJECTIVE: The objective of this work was to identify the mechanisms of programmed cell death and inflammasome components in human oral mucosal lesions of paracoccidioidomycosis through immunohistochemical methods and identification of RIPK-3, IL1ß, IL18, NLRP-1 and caspase-1. Thirty specimens were included, and a histopathological analysis of the lesions was performed using haematoxylin-eosin staining. RESULTS: Our results on in situ expression of inflammasome elements and programmed cell death showed increased expression of IL-1ß, NLRP-1, caspase-1 and RIPK-3. We suggest that inflammasome complex participate in the immunopathogenesis in paracoccidioidomycosis oral lesions in an interplay with RIPK3.

Inflammasome and Inflammatory Programmed Cell Death in Chromoblastomycosis.

Chromoblastomycosis (CBM) is a chronic, progressive fungal disease of the skin and subcutaneous tissue caused by a group of dematiaceous fungi. Verrucous lesions present parasite-rich granulomas and predominance of a Th2 patterns of cytokines. The inflammasome constitutes a macromolecular protein complex that play a role in the activation of caspase 1 that cleaves pro-IL1ß and pro-IL18, essential mediators of inflammation, and also activates pyroptosis. We intended to explore the presence and a possible role of inflammasome elements in cutaneous human lesions in CBM, considering the expression of IL1ß, IL18, caspase 1, NLRP1, and also RIPK3, a key downstream component of necroptosis signaling. 35 skin biopsies of cutaneous lesions of verrucous form of CBM and 10 biopsies from normal skin were selected. The diagnosis was based on histological and clinical analysis. An immunohistochemical protocol was performed. The histopathological analysis evidenced epidermis with hyperkeratosis, irregular acanthosis, and micro abscesses. The dermis presented suppurative granulomas and inflammatory infiltrate composed by giant cells, macrophages, epithelioid cells, lymphocytes, and some eosinophils. Positive cells were distributed in the inflammatory infiltrate, with an increased number of cells expressing caspase 1, IL1ß and IL18. Cells expressing RIPK3 and NLRP1 were less frequent. The intense presence of caspase 1, IL1ß and IL18, allied to NLRP1 expression, suggest that inflammasome and pyroptosis could play a role in the immune response against fungal agents of CBM. Our results, allied to data from literature, could suggest that inflammasome-mediated response and pyroptosis could be a target to be explored to decrease CBM lesions.

Imbalanced IL-1B and IL-18 Expression in Sézary Syndrome.

Sézary syndrome (SS) is a rare and aggressive type of cutaneous T-cell lymphoma, with an abnormal inflammatory response in affected skin. The cytokines IL-1B and IL-18, as key signaling molecules in the immune system, are produced in an inactive form and cleave to the active form by inflammasomes. In this study, we assessed the skin, serum, peripheral mononuclear blood cell (PBMC) and lymph-node samples of SS patients and control groups (healthy donors (HDs) and idiopathic erythroderma (IE) nodes) to investigate the inflammatory markers IL-1B and IL-18 at the protein and transcript expression levels, as potential markers of inflammasome activation. Our findings showed increased IL-1B and decreased IL-18 protein expression in the epidermis of SS patients; however, in the dermis layer, we detected increased IL-18 protein expression. In the lymph nodes of SS patients at advanced stages of the disease (N2/N3), we also detected an enhancement of IL-18 and a downregulation of IL-1B at the protein level. Moreover, the transcriptomic analysis of the SS and IE nodes confirmed the decreased expression of IL1B and NLRP3, whereas the pathway analysis indicated a further downregulation of IL1B-associated genes. Overall, the present findings showed compartmentalized expressions of IL-1B and IL-18 and provided the first evidence of their imbalance in patients with Sézary syndrome.

Plasmacytoid Dendritic Cells, the Expression of the Stimulator of Interferon Genes Protein (STING) and a Possible Role of Th17 Immune Response in Cervical Lesions Mediated by Human Papillomavirus.

Human papillomavirus (HPV) is a virus with a DNA structure that specifically targets squamous epithelial cells. In individuals with a healthy immune system, HPV infection is typically resolved naturally, leading to spontaneous regression. However, when the viral genetic material integrates into the host DNA, it can disrupt the immune response and eventually give rise to neoplastic manifestations. Remarkably, HPV infection appears to activate a protein called Stimulator of Interferon genes (STING), which contributes to the infiltration of Treg Foxp3 + cells. This cellular response acts as a predisposing factor in patients with HPV, potentially exacerbating the progression of the infection. The STING is versatile in different circumstances and can play a role in the immune response as an anti-tumour therapeutic target in HPV-related carcinogenesis. The function of Th17 cells is ambiguous, depending on the microenvironment in the tumour. In this study, 46 biopsies of the uterine cervix of human immunodeficiency virus (HIV) positive patients were divided into three groups: I-cervicitis (10); II-low-grade intraepithelial neoplasia (26); III-moderate or severe intraepithelial neoplasia (10) and it was performed an immunohistochemical technique with the specific antibodies to HPV, CD123, STING and IL17. Immunostained cells were quantified and statistically analysed. Antigens of HPV were detected in the cervical intraepithelial neoplasia (CIN) groups and were absent in cervicitis group. The expression of CD123 was positive in 10.87% of the casuistic, with no statistical difference among groups. STING was present in the three groups. Group 1 presented an area fraction that varied from 3 to 20%, group 2 presented a variation of 3-23% and group 3 presented an area fraction between 4 and 12%. Cells expressing IL17 were present in three groups, more frequent in cervicitis. Considering that the casuistic is composed of women carrying HIV, this infectious agent could influence the numerical similarities of the cells studied among three groups, even in the absence of HPV.

Stimulator of Interferon genes protein (STING) is versatile in different mechanisms in the immune response and may be a target in HPV-related carcinogenesis. Through immunohistochemistry it was detected IL17 in cervicitis and lesions with HPV. Also, CD123 + plasmacytoid cells seems to play a role both in cervicitis and lesions mediated by HPV.

Livedoid vasculopathy in 75 Brazilian patients in a single-center institution: Clinical, histopathological and therapy evaluation.

This study presents a single center experience with livedoid vasculopathy (LV). A rare disease that can lead to severe quality of life impairment. Characterize clinical data of LV patients at the Dermatology Division at the University of São Paulo. A retrospective and transversal study was conducted, from 1 January 2005 to 31 December 2019. About 75 patients diagnosed as LV and confirmed by skin biopsy were included. Epidemiology, clinical appearance, histopathology data, and treatment history were observed. There were 78.66% Caucasian women, with a mean age of 39.9 years. Frequent cutaneous manifestations were ulcers, atrophic blanche-like scars, hyperpigmentation, purpuras, telangiectasias, and livedo racemosa. Pain, pruritus, and hypoesthesia were the main symptoms. After treatment, almost 40% of cases relapsed during spring and summer months. About 66% of cases had thrombophilia factors associated, such as high levels of lipoprotein(a). Frequent treatments included acetylsalicylic acid, pentoxifylline, and diosmin with hesperidin. Not being a prospective study. This research provides useful data on Latin American LV patients, indicating multifactorial conditions involved in LV pathogenesis. An extensive work-up including autoimmune laboratory tests, thrombophilia factors, and other conditions associated with venous stasis should be part of LV investigation and controlled to improve treatment response.

Proinflammatory and regulatory mechanisms in allergic contact dermatitis caused by methylchloroisothiazolinone and methylisothiazolinone.

BACKGROUND: Methylchloroisothiazolinone (MCI) and methylisothiazolinone (MI) are the cause of an increasing number of contact allergies. Understanding the mechanisms by which MCI/MI induces proinflammatory and regulatory factors production is necessary to understand the outcome of allergic contact dermatitis (ACD). OBJECTIVES: To evaluate the dysfunction of proinflammatory cytokines and regulatory factors in the positive MCI/MI patch test at the transcriptional and protein expression levels. Moreover, to analyse the cytokines production induced by MI in peripheral blood mononuclear cells (PBMCs). MATERIALS AND METHODS: The selected patients had positive MCI/MI patch test results. The expression of proinflammatory factors was evaluated by q-PCR and immunochemistry at 48 hours of positive MCI/MI patch test. The MCI/MI- or MI- induced secretion of IL-1ß, TNF and IL-6 by PBMC was analysed by flow cytometry. RESULTS: The results showed a decreased TLR4 expression with upregulated IL6, FOXP3, IL10 and TGFß mRNA expression as assessed by q-PCR at the site of the MCI/MI skin reaction. We detected increased protein levels of TLR4, FOXP3 and IL-10 in the dermis layer in the ACD reaction by immunocitochemistry. Moreover, MCI/MI induced proinflammatory cytokine production by PBMC through the NF-κB signalling pathway. CONCLUSION: Considering the altered innate immune response triggered by MCI/MI sensitization, these findings indicate that the regulatory process at the induction phase of ACD is a crucial mechanism. Given the increase in occupational and domestic exposure to MCI/MI, the underlying immunological mechanisms should be understood.

M2-Polarized Macrophages Determine Human Cutaneous Lesions in Lacaziosis.

Lacaziosis is a cutaneous chronic mycosis caused by Lacazia loboi. Macrophages are important cells in the host immune response in fungal infections. The macrophage population exhibits strong plasticity that varies according to the stimuli in the microenvironment of lesions M1 profile promotes a Th1 pattern of cytokines and a microbicidal function and M2 is related to Th2 cytokines and immunomodulatory response. We investigated the population of M1 and M2 polarized macrophages in human cutaneous lesions. A total of 27 biopsies from human lesions were submitted to an immunohistochemistry protocol using antibodies to detect M1 and M2 macrophages (Arginase-1, CD163, iNOS, RBP-J and cMAF). We could observe high number of cells expressing Arginase1, CD163 and c-MAF that correspond to elements of the M2 profile of macrophage, over iNOS and RBP-J (elements of the M1 profile). The results suggest a predominant phenotype of M2 macrophages, which have an immunomodulatory role and probably contributing to chronicity of Lacaziosis.

IL-37 and leprosy: A novel cytokine involved in the host response to Mycobacterium leprae infection.

Leprosy is a chronic infectious granulomatous disease caused by Mycobacterium leprae, in which the clinical outcome depends on the pattern of the host immune response. Because it is a spectral disease, leprosy is a good model for studying the immunology of the pathogen-host relationship. Although previous studies have characterized the participation of cytokine profiles such as Th1, Th2, Th7, Treg, Th9, and Th22 responses in leprosy, the role of new cytokines such as IL-37 have not yet been described for the spectral model of the disease. Here, we used an immunohistochemical technique to evaluate IL-37 expression in the skin of patients with leprosy. The expression of this cytokine was observed in the keratinocytes, endothelial cells, macrophages, and lymphocytes. Moreover, the IL-37 expression level was increased in patients with the tuberculoid (TT) form when compared to those with the lepromatous leprosy (LL) form in keratinocytes, endotheliocytes, and lymphocytes. However, in the macrophages, the cytokine expression was more intense in the LL form of the disease. These results point to the effective participation of IL-37 in the immunopathogenesis of leprosy, which is expressed in both the epidermal cells and the dermis.

Immunohistochemical characterization of the M4 macrophage population in leprosy skin lesions.

BACKGROUND: Since macrophages are one of the major cell types involved in the Mycobacterium leprae immune response, roles of the M1 and M2 macrophage subpopulations have been well defined. However, the role of M4 macrophages in leprosy or other infectious diseases caused by mycobacteria has not yet been clearly characterized. This study aimed to investigate the presence and potential role of M4 macrophages in the immunopathology of leprosy. METHODS: We analyzed the presence of M4 macrophage markers (CD68, MRP8, MMP7, IL-6, and TNF-α) in 33 leprosy skin lesion samples from 18 patients with tuberculoid leprosy and 15 with lepromatous leprosy by immunohistochemistry. RESULTS: The M4 phenotype was more strongly expressed in patients with the lepromatous form of the disease, indicating that this subpopulation is less effective in the elimination of the bacillus and consequently is associated with the evolution to one of the multibacillary clinical forms of infection. CONCLUSION: M4 macrophages are one of the cell types involved in the microbial response to M. leprae and probably are less effective in controlling bacillus replication, contributing to the evolution to the lepromatous form of the disease.

Autologous hematopoietic stem cell transplant in a patient with leprosy: Is it safe?

A patient with non-Hodgkin lymphoma, preparing for an autologous hematopoietic stem cell transplant (HSCT), developed leprosy. The patient was successfully treated with rifampicin, ofloxacin, and doxycycline, and the HSCT was performed without complications, being the first report, to our knowledge, of leprosy in an autologous HSCT patient.

The cytotoxic T cells may contribute to the in situ immune response in Jorge Lobo's Disease human lesions.

Jorge Lobo's Disease (JLD) is a cutaneous chronic granulomatous disease caused by the pathogenic fungus Lacazia loboi. It is characterized by a granulomatous reaction with multinucleated giant cells and high number of fungal cells. In order to contribute to the comprehension of immune mechanisms in JLD human lesions, we studied the cytotoxic immune response, focusing on TCD8+ and NK cells, and granzyme B. Forty skin biopsies of lower limbs were selected and an immunohistochemistry protocol was developed to detect CD8+ T cells, NK cells and Granzyme B. In order to compare the cellular populations, we also performed a protocol to visualize TCD4+ cells. Immunolabeled cells were quantified in nine randomized fields in the dermis. Lesions were characterized by inflammatory infiltrate of macrophages, lymphocytes, epithelioid and multinucleated giant cells with intense number of fungal forms. There was a prevalence of CD8 over CD4 cells, followed by NK cells. Our results suggest that in JLD the cytotoxic immune response could represent another important mechanism to control Lacazia loboi infection. We may suggest that, although CD4+ T cells are essential for host defense in JLD, CD8+ T cells could play a role in the elimination of the fungus.

Th17 and regulatory T cells contribute to the in situ immune response in skin lesions of Jorge Lobo's disease.

Jorge Lobo's disease (JLD) is a chronic granulomatous mycosis described in various Latin American countries. The main objective of the present study was to investigate the possible role of Th17 and Foxp3+ Treg cells in the pathogenesis of Jorge Lobo's disease. Human skin biopsies were submitted to an immunohistochemistry protocol to detect Foxp3, interleukin (IL)-1beta, CD25, IL-6, IL-17, and IL-23. The epidermis presented acanthosis, hyperkeratosis, and frequent presence of fungi. The dermis presented inflammatory infiltrate comprising macrophages, lymphocytes, epithelioid and multinucleated cells, and an intense number of fungi. Foxp3+ Treg cells and IL-17+ cells were visualized in lymphocytes in the inflammatory infiltrate. IL-1, IL-2R (CD25), IL-6, and IL-23 were visualized in the dermis, intermingled with fungal cells, permeating or participating of the granuloma. Following IL-17, the most prominent cytokine was IL-6. IL-23 and cells expressing CD25 were present in fewer number. The comparative analysis between IL-17 and Foxp3 demonstrated a statistically significant increased number of IL-17+ cells. Th17 cells play a role in the immune response of JLD. IL-1beta and IL-6 added to the previously described increased number of TGF-beta would stimulate such pattern of response. Th17 cells could be present as an effort to modulate the local immune response; however, high levels of a Th17 profile could overcome the role of Treg cells. The unbalance between Treg/Th17 cells seems to corroborate with the less effective immune response against the fungus.

Up-regulation of Proinflammatory Genes and Cytokines Induced by S100A8 in CD8+ T Cells in Lichen Planus.

Lichen planus (LP) is a chronic inflammatory mucocutaneous disease. The inflammatory status of LP may be related to S100A8 (myeloid-related protein 8; MRP8) activation of cytotoxic cells. The aims of this study were to evaluate S100A8 expression in skin lesions and the in vitro effects of S100A8 on CD8+ T cells and natural killer (NK) cells in LP. Increased levels of S100A8/S100A9 were detected in the skin lesions as well as in the sera of subjects with LP. S100A8 expression induced an increased cytotoxic response by peripheral blood CD8+CD107a+ T cells as well as by NK CD56bright cells in patients with LP. Increased expression of interleukin (IL)-1ß, tumour necrosis factor (TNF) and IL-6 in the CD8+ T cells of patients with LP was induced by S100A8, in contrast to the control group that produced IL- 10 and interferon (IFN) type I genes. These data suggest that, in individuals with LP, S100A8 may exert distinct immunomodulatory and cytotoxicity functions.

Post-kala-azar dermal leishmaniasis and leprosy: case report and literature review.

BACKGROUND: Post-kala-azar dermal leishmaniasis (PKDL) is a dermal complication of visceral leishmaniasis (VL), which may occur after or during treatment. It has been frequently reported from India and the Sudan, but its occurrence in South America has been rarely reported. It may mimic leprosy and its differentiation may be difficult, since both diseases may show hypo-pigmented macular lesions as clinical presentation and neural involvement in histopathological investigations. The co-infection of leprosy and VL has been reported in countries where both diseases are endemic. The authors report a co-infection case of leprosy and VL, which evolved into PKDL and discuss the clinical and the pathological aspects in the patient and review the literature on this disease. CASE PRESENTATION: We report an unusual case of a 53-year-old female patient from Alagoas, Brazil. She presented with leprosy and a necrotizing erythema nodosum, a type II leprosy reaction, about 3 month after finishing the treatment (MDT-MB) for leprosy. She was hospitalized and VL was diagnosed at that time and she was successfully treated with liposomal amphotericin B. After 6 months, she developed a few hypo-pigmented papules on her forehead. A granulomatous inflammatory infiltrate throughout the dermis was observed at histopathological examination of the skin biopsy. It consisted of epithelioid histiocytes, lymphocytes and plasma cells with the presence of amastigotes of Leishmania in macrophages (Leishman's bodies). The diagnosis of post-kala-azar dermal leishmaniasis was established because at this time there was no hepatosplenomegaly and the bone marrow did not show Leishmania parasites thus excluding VL. About 2 years after the treatment of PKDL with liposomal amphotericin B the patient is still without PKDL lesions. CONCLUSION: Post-kala-azar dermal leishmaniasis is a rare dermal complication of VL that mimics leprosy and should be considered particularly in countries where both diseases are endemic. A co-infection must be seriously considered, especially in patients who are non-responsive to treatment or develop persistent leprosy reactions as those encountered in the patient reported here.

Evaluation of the 2008 World Health Organization classification for non-mycosis fungoides, non-Sezary syndrome T/NK-cell lymphomas with primary cutaneous involvement.

BACKGROUND: Cutaneous non-mycosis fungoides non-Sezary syndrome T/NK cell lymphomas (non-MF/non-SS CTCL) are rare. In 2005, a consensus of the World Health Organization (WHO) and European Organization for Research and Treatment of Cancer (EORTC) classifications for primary cutaneous lymphomas was established. These guidelines were then adopted into the WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, 2008. This study aims to assess the applicability of the WHO 2008 classification in a retrospective series of CTCL cases registered in a reference academic center in Brazil. METHODS: Twenty-seven patients with non-MF/non-SS CTCL were studied. Clinical, histopathological and immunophenotypical features based on an extensive panel of antibodies were applied to classify the cases according to the WHO, 2008. RESULTS: Overall, diagnostic categories included eight (29.6%) cutaneous anaplastic large-cell lymphoma, five (18.5%) lymphomatoid papulosis, six (22.2%) extranodal natural killer (NK)/T-cell lymphoma, nasal type, five (18.5%) adult T-cell leukemia/lymphoma, one (3.7%) cutaneous CD8-positive aggressive epidermotropic cytotoxic T-cell lymphoma and two (7.4%) of peripheral T-cell lymphoma not otherwise specified (NOS). CONCLUSIONS: The WHO classification (2008) was applicable to most cases of non-MF/non-SS CTCL, while some cases remained unclassified and were considered NOS-peripheral T-cell lymphoma. An unexpected high frequency of NK/T-cell lymphoma nasal type was observed.

Jorge Lobo's disease: immunohistochemical characterization of dendritic cells in cutaneous lesions.

BACKGROUND: Jorge Lobo's disease (JLD) is a cutaneous chronic mycosis caused by Lacazia loboi. We studied Factor XIIIa + dermal dendrocytes (FXIIIa + DD), Langerhans cells (LC) through the expression of langerin and the expression of S100 protein. METHODS: A total of 41 biopsies and 10 normal skins (control) were developed with a polymer-based immunohistochemical method. RESULTS: Lesions presented infiltrate comprising macrophages, some asteroid corpuscles, lymphocytes, multinucleated giant cells and a large number of fungi. LCs presented short dendrites and were scarcely distributed. Dermal langerin + cells were detected in nine JLD lesions. FXIIIa + DD were hypertrophic, visualized in the inflammatory infiltrate of JLD lesions. Cells S100+ were present in JLD and control group with a similar number of cells. A total of 14 specimens did not express FXIIIa, and this considerable number probably contributed to the statistical similarity with the control group. CONCLUSIONS: The results indicate that LCs are present in the immune response against Lacazia loboi. Some dermal langerin + cells could be another subset of dendritic cells. Our data indicate changes of LCs in JLD cutaneous lesions and present, for the first time, results that show langerin + cells in the dermis and corroborate previous observations on the participation of FXIIIa + DD in the in situ immune response in JLD.

Plasmacytoid dendritic cells in cutaneous lesions of patients with chromoblastomycosis, lacaziosis, and paracoccidioidomycosis: a comparative analysis.

Plasmacytoid dendritic cells (pDCs) are characterized by expression of CD123 and BDCA-2 (Blood Dendritic Cell Antigen 2) (CD303) molecules, which are important in innate and adaptive immunity. Chromoblastomycosis (CBM), lacaziosis or Jorge Lobo's disease (JLD), and paracoccidioidomycosis (PCM), are noteworthy in Latin America due to the large number of reported cases. The severity of lesions is mainly determined by the host's immune status and in situ responses. The dendritic cells studied in these fungal diseases are of myeloid origin, such as Langerhans cells and dermal dendrocytes; to our knowledge, there are no data for pDCs. Forty-three biopsies from patients with CBM, 42 from those with JLD and 46 diagnosed with PCM, were evaluated by immunohistochemistry. Plasmacytoid cells immunostained with anti-CD123 and anti-CD303 were detected in 16 cases of CBM; in those stained with anti-CD123, 24 specimens were obtained from PCM. We did not detect the presence of pDCs in any specimen using either antibody in JLD. We believe that, albeit a secondary immune response in PCM and CBM, pDCs could act as a secondary source of important cytokines. The BDCA-2 (CD303) is a c-type lectin receptor involved in cell adhesion, capture, and processing of antigens. Through the expression of the c-lectin receptor, there could be an interaction with fungi, similar to other receptors of this type, namely, CD207 in PCM and CD205 and CD209 in other fungal infections. In JLD, the absence of expression of CD123 and CD303 seems to indicate that pDCs are not involved in the immune response.

Methotrexate for refractory adult atopic dermatitis leads to alterations in cutaneous IL-31 and IL-31RA expression.

BACKGROUND: Methotrexate (MTX) is an alternative treatment for patients with moderate/severe atopic dermatitis (AD). OBJECTIVE: The authors evaluated the effect of MTX on the cutaneous expression of cytokines and chemokines that are involved in the inflammatory response in adult AD patients who received treatment with methotrexate for 24 weeks. METHODS: The authors conducted a prospective single-institution cohort study with 12 adults with moderate/severe AD who received oral MTX (15 mg/wk for 24 wks) and 10 non-atopic matched controls. The comparison was made of skin biopsies of lesional and non-lesional skin, pre- and post MTX treatment. The authors analyzed mean epidermal thickness and expression of IL-31, IL-31RA, OSMR, TSLP, Ki67, IL-4 mRNA, IL-6, IL-10, TNF-α, IFN-γ, TARC, and CCL-22. RESULTS: There was a reduction in mean epidermal thickness (p = 0.021), an increase in IL-31RA expression (immunohistochemistry) in the epidermis (p = 0.016) and a decrease in IL-31 gene expression (p = 0.019) on lesional AD skin post-MTX treatment. No significant changes in the cutaneous expression of the other evaluated markers were identified. STUDY LIMITATIONS: Small sample size and limited length of follow-up. CONCLUSIONS: Treatment with MTX in adults with moderate/severe AD reduced epidermal hyperplasia and changed the cutaneous expression of inflammatory cytokines and receptors that are mainly related to pruritus, including IL-31 and IL-31RA. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03327116.