Ex vivo culture of lesional psoriasis skin for pharmacological testing

Background: Psoriasis is a chronic, inflammatory skin disorder resulting from a complex interplay between immune and skin cells via release of soluble mediators. While a lot is known about the molecular mechanisms behind psoriasis pathogenesis, there is still a need for preclinical research models that accuratelyreplicate the disease. Objective: This study aimed to develop and characterize ex vivo culture of psoriasis skin as a model for pharmacological testing, where the immunological events of psoriasis can be followed. Methods: Full thickness punch biopsies of lesional psoriasis skin were cultured in submerged conditions up to 144 h followingin situ T cell stimulation with rhIL-23 and anti-CD3 and anti-CD28 antibodies. The Tcell mediated skin inflammation was assessed by gene and protein l analysis for a panel of inflammatory mediators. Tissue integrity and morphology were evaluated by histological analysis. Results: T cell stimulation resulted in functional and psoriasis specificin situ activation of T cells. The expression levels of most of the proinflammatory mediators related to both immune and skin cells were comparable to these in freshly isolated tissue at 48 and 96 h of culture. Tissue integrity and morphology were sustained up to 96 h. Treatment with a corticosteroid reduced the expression of several proinflammatory cytokines and chemokines, whereas anti-IL-17A antibody treatment reduced the expression of the IL-17A downstream markers IL-8 and DEFB4. Conclusion: By preserving keyimmunopathological mechanisms of psoriasis, ex vivo culture of psoriasis skin can be used for the investigation of inflammatory processes of psoriasis and for preclinical drug discovery research

Human CLA+ memory T cell and cytokines in psoriasis

Psoriasis is a common inflammatory skin condition resulting from the interplay between epidermal keratinocytes and immunological cellular components. This sustained inflammation is essentially driven by pro-inflammatory cytokines with the IL-23/IL-17 axis playing a critical central role, as proved by the clinical efficacy of their blockade in patients. Among all the CD45R0+ memory T cell subsets, those with special tropism for cutaneous tissues are identified by the expression of the Cutaneous Lymphocyte-associated Antigen (CLA) carbohydrate on their surface, that is induced during T cell maturation particularly in the skin-draining lymph nodes. Because of their ability to recirculate between the skin and blood, circulating CLA+ memory T cells reflect the immune abnormalities found in different human cutaneous conditions, such as psoriasis. Based on this premise, studying the effect of different environmental microbial triggers and psoriatic lesional cytokines on CLA+ memory T cells, in the presence of autologous epidermal cells from patients, revealed important IL-17 cytokines responses that are likely to enhance the pro-inflammatory loop underlying the development of psoriatic lesions. The goal of this mini-review is to present latest data regarding cytokines implicated in plaque and guttate psoriasis immunopathogenesis from the prism of CLA+ memory T cells, that are specifically related to the cutaneous immune system

Arginine transport is impaired in C57Bl/6 mouse macrophages as a result of a deletion in the promoter of slc7a2 (CAT2) and Leishmania infection is reduced

Host genetic factors play a crucial role in immune response. To determine whether the differences betweenC57Bl/6 and BALB-C mice are due only to the production of cytokines by T-helper 1 cells or T-helper 2 cells,we obtained bone marrow–derived macrophages from both strains and incubated them with these cytokines.Although the induction of Nos2 and Arg1 was similar in the 2 strains, infectivity to Leishmania major differed,as did macrophage uptake of arginine, which was higher in BALB-C macrophages. The levels of interferon γ–and interleukin 4–dependent induction of the cationic amino acid transporter SLC7A2 (also known as “cationicamino acid transporter 2,” or “CAT2”) were decreased in macrophages from C57Bl/6 mice. This reductionwas a result of a deletion in the promoter of one of the 4 AGGG repeats. These results demonstrate that theavailability of arginine controls critical aspects of macrophage activation and reveal a factor for susceptibility to Leishmania infection

Allergen sensitization stratifies IL-31 production by memory T cells in atopic dermatitis patients

Background:The role of allergen sensitization in IL-31 production by T cells and specifically in the clinical context of atopic dermatitis (AD) has not been characterized. MethodsThe response to house dust mite (HDM) in purified memory T cells cocultured with epidermal cells from AD patients (n=58) and control subjects (n=11) was evaluated. AD-associated cytokines from culture supernatants, plasma proteins and mRNA expression from cutaneous lesions were assessed and related with the clinical features of the patients. ResultsHDM-induced IL-31 production by memory T cells defined two subsets of AD patients according to the presence or absence of IL-31 response. Patients in the IL-31 producing group showed a more inflammatory profile, and increased HDM-specific (sp) and total IgE levels compared to the IL-31 non-producing group. A correlation between IL-31 production and patient's pruritus intensity, plasma CCL27 and periostin was detected. When the same patients were analyzed based on sp IgE and total IgE levels, an increased IL-31 in vitro response, as well as type 2 markers in plasma and cutaneous lesions, was found in patients with sp IgE levels > 100 kUA/L and total IgE levels > 1000 kU/L. The IL-31 response by memory T cells was restricted to the cutaneous lymphocyte-associated antigen (CLA)(+) T-cell subset. ConclusionIgE sensitization to HDM allows stratifying IL-31 production by memory T cells in AD patients and relating it to particular clinical phenotypes of the disease

Pharmacologic modulation of RORγt translates to efficacy in preclinical and translational models of psoriasis and inflammatory arthritis.

The IL-23/IL-17 pathway is implicated in autoimmune diseases, particularly psoriasis, where biologics targeting IL-23 and IL-17 have shown significant clinical efficacy. Retinoid-related orphan nuclear receptor gamma t (RORγt) is required for Th17 differentiation and IL-17 production in adaptive and innate immune cells. We identified JNJ-54271074, a potent and highly-selective RORγt inverse agonist, which dose-dependently inhibited RORγt-driven transcription, decreased co-activator binding and promoted interaction with co-repressor protein. This compound selectively blocked Th17 differentiation, significantly reduced IL-17A production from memory T cells, and decreased IL-17A- and IL-22-producing human and murine γδ and NKT cells. In a murine collagen-induced arthritis model, JNJ-54271074 dose-dependently suppressed joint inflammation. Furthermore, JNJ-54271074 suppressed IL-17A production in human PBMC from rheumatoid arthritis patients. RORγt-deficient mice showed decreased IL-23-induced psoriasis-like skin inflammation and cytokine gene expression, consistent with dose-dependent inhibition in wild-type mice through oral dosing of JNJ-54271074. In a translational model of human psoriatic epidermal cells and skin-homing T cells, JNJ-54271074 selectively inhibited streptococcus extract-induced IL-17A and IL-17F. JNJ-54271074 is thus a potent, selective RORγt modulator with therapeutic potential in IL-23/IL-17 mediated autoimmune diseases

CLA+ T cell response to microbes in psoriasis

Streptococcus pyogenes throat infection is a clinically relevant trigger of both guttate and chronic plaque psoriasis, and it provides an ideal context in which to study the pathogenesis of these diseases using an antigen-dependent approach. Circulating cutaneous lymphocyte-associated antigen (CLA) positive (+) memory T cells are a subset of peripheral lymphocytes whose phenotype and function are related to immunological mechanisms in the skin. These cells are considered peripheral biomarkers of T-cell-mediated skin diseases. The coculture of autologous epidermal cells with CLA+ T cells from psoriasis patients activated by S. pyogenes allows the reproduction of the ex vivo initial molecular events that occur during psoriatic lesion formation. With cooperation of autologous epidermal cells, S. pyogenes selectively activates CLA+ T cells both in guttate and plaque psoriasis, inducing key mediators, including an IL-17 response. Here, we explore potential new mechanisms of psoriasis development including the influence of HLA-Cw6 on S. pyogenes CLA+ T cell activation in guttate psoriasis, the relevance of IL-9 on microbe induced IL-17 response in guttate and plaque psoriasis, and novel effector functions of Candida albicans. This review will summarize recent knowledge of psoriatic mechanisms elicited by microbes that have been studied through an innovative translational perspective based on CLA+ T cell-mediated cutaneous immune response

CLA+ memory T cells in atopic dermatitis: CLA+ T cells and atopic dermatitis

Circulating skin-homing cutaneous lymphocyte-associated antigen (CLA)+ T cells constitute a small subset of human memory T cells involved in several aspects of atopic dermatitis: Staphylococcus aureus related mechanisms, the abnormal Th2 immune response, biomarkers, clinical aspects of the patients, pruritus, and the mechanism of action of targeted therapies. Superantigens, IL-13, IL-31, pruritus, CCL17 and early effects on dupilumab-treated patients have in common that they are associated with the CLA+ T cell mechanisms in atopic dermatitis patients. The function of CLA+ T cells corresponds with the role of T cells belonging to the skin-associated lymphoid tissue and could be a reason why they reflect different mechanisms of atopic dermatitis and many other T cell mediated skin diseases. The goal of this review is to gather all this translational information of atopic dermatitis pathology

Streptococcus Induces Circulating CLA+ Memory T-Cell-Dependent Epidermal Cell Activation in Psoriasis

Streptococcal throat infection is associated with a specific variant of psoriasis and with HLA-Cw6 expression. In this study, activation of circulating psoriatic cutaneous lymphocyte–associated antigen (CLA)+ memory T cells cultured together with epidermal cells occurred only when streptococcal throat extracts were added. This triggered the production of Th1, Th17, and Th22 cytokines, as well as epidermal cell mediators (CXCL8, CXCL9, CXCL10, and CXCL11). Streptococcal extracts (SEs) did not induce any activation with either CLA− cells or memory T cells cultured together with epidermal cells from healthy subjects. Intradermal injection of activated culture supernatants into mouse skin induced epidermal hyperplasia. SEs also induced activation when we used epidermal cells from nonlesional skin of psoriatic patients with CLA+ memory T cells. Significant correlations were found between SE induced upregulation of mRNA expression for ifn-γ, il-17, il-22, ip-10, and serum level of antistreptolysin O in psoriatic patients. This study demonstrates the direct involvement of streptococcal infection in pathological mechanisms of psoriasis, such as IL-17 production and epidermal cell activation

Pharmacologic modulation of RORγt translates to efficacy in preclinical and translational models of psoriasis and inflammatory arthritis.

The IL-23/IL-17 pathway is implicated in autoimmune diseases, particularly psoriasis, where biologics targeting IL-23 and IL-17 have shown significant clinical efficacy. Retinoid-related orphan nuclear receptor gamma t (RORγt) is required for Th17 differentiation and IL-17 production in adaptive and innate immune cells. We identified JNJ-54271074, a potent and highly-selective RORγt inverse agonist, which dose-dependently inhibited RORγt-driven transcription, decreased co-activator binding and promoted interaction with co-repressor protein. This compound selectively blocked Th17 differentiation, significantly reduced IL-17A production from memory T cells, and decreased IL-17A- and IL-22-producing human and murine γδ and NKT cells. In a murine collagen-induced arthritis model, JNJ-54271074 dose-dependently suppressed joint inflammation. Furthermore, JNJ-54271074 suppressed IL-17A production in human PBMC from rheumatoid arthritis patients. RORγt-deficient mice showed decreased IL-23-induced psoriasis-like skin inflammation and cytokine gene expression, consistent with dose-dependent inhibition in wild-type mice through oral dosing of JNJ-54271074. In a translational model of human psoriatic epidermal cells and skin-homing T cells, JNJ-54271074 selectively inhibited streptococcus extract-induced IL-17A and IL-17F. JNJ-54271074 is thus a potent, selective RORγt modulator with therapeutic potential in IL-23/IL-17 mediated autoimmune diseases