RESUMEN
BACKGROUND: Atopic dermatitis (AD), a chronic inflammatory skin disease with T cell activation as a key feature, in which Th2 cell-mediated responses play a pivotal role. Regulatory T cells (Treg) are central immune cells that restrict autoimmunity and inflammation in the body. Patients with immune dysregulation, polyendocrinopathy, or enteropathy X-linked syndrome, an immune disease characterized by a deficiency in Treg, develop skin inflammation and allergic disorders, indicating that Treg play a crucial role in the development of allergic skin inflammation. OBJECTIVE: we investigated the underlying mechanisms by which Treg control cutaneous allergic inflammation. METHODS: An allergic skin inflammation mouse model was constructed using MC903, and Treg-depleted mouse model was constructed using diphtheria toxin. Neutralization of IFN-γ was constructed using anti-mouse-IFN-γ mouse antibody. Neutrophil infiltration was analyzed by flow cytometry and immunohistochemistry. Neutrophil extracellular traps (NETs), a process called NETosis, were detected using immunofluorescence. In vitro neutrophil stimulation and immunocytochemistry was conducted to demonstrate the effect of IFN-γ on NETosis. RESULTS: The depletion of Foxp3+ Treg led to significantly exacerbated AD-like skin inflammation, including increased recruitment of neutrophils and expression of Th1 cytokine IFN-γ. Neutrophil infiltrating in skin of Treg-depleted mice released more NETs than wild type. Neutralization of IFN-γ abolished neutrophil infiltration and NETosis in Treg-depleted mice. Neutrophils stimulated with IFN-γ were more prone to release NETs in vitro. Finally, Foxp3+ Treg control cutaneous allergic inflammation by regulating IFN-γ-driven neutrophilic infiltration and NETosis. CONCLUSION: Our results highlight the previously underestimated Treg-IFN-γ-neutrophil inflammatory axis.
Asunto(s)
Dermatitis Atópica , Modelos Animales de Enfermedad , Trampas Extracelulares , Factores de Transcripción Forkhead , Interferón gamma , Infiltración Neutrófila , Neutrófilos , Piel , Linfocitos T Reguladores , Animales , Linfocitos T Reguladores/inmunología , Interferón gamma/metabolismo , Interferón gamma/inmunología , Factores de Transcripción Forkhead/metabolismo , Ratones , Trampas Extracelulares/inmunología , Dermatitis Atópica/inmunología , Dermatitis Atópica/patología , Dermatitis Atópica/inducido químicamente , Piel/inmunología , Piel/patología , Infiltración Neutrófila/inmunología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Humanos , Ratones Endogámicos C57BL , FemeninoRESUMEN
Dendritic cells (DCs) are readily generated from the culture of mouse bone marrow (BM) treated with either granulocyte macrophage-colony stimulating factor (GM-CSF) or FMS-like tyrosine kinase 3 ligand (FLT3L). CD11c+MHCII+ or CD11c+MHCIIhi cells are routinely isolated from those BM cultures and generally used as in vitro-generated DCs for a variety of experiments and therapies. Here, we examined CD11c+ cells in the BM culture with GM-CSF or FLT3L by staining with a monoclonal antibody 2A1 that is known to recognize mature or activated DCs. Most of the cells within the CD11c+MHCIIhi DC gate were 2A1+ in the BM culture with GM-CSF (GM-BM culture). In the BM culture with FLT3L (FL-BM culture), almost of all the CD11c+MHCIIhi cells were within the classical DC2 (cDC2) gate. The analysis of FL-BM culture revealed that a majority of cDC2-gated CD11c+MHCIIhi cells exhibited a 2A1-CD83-CD115+CX3CR1+ phenotype, and the others consisted of 2A1+CD83+CD115-CX3CR1- and 2A1-CD83-CD115-CX3CR1- cells. According to the antigen uptake and presentation, morphologies, and gene expression profiles, 2A1-CD83-CD115-CX3CR1- cells were immature cDC2s and 2A1+CD83+CD115-CX3CR1- cells were mature cDC2s. Unexpectedly, however, 2A1-CD83-CD115+CX3CR1+ cells, the most abundant cDC2-gated MHCIIhi cell subset in FL-BM culture, were non-DCs. Adoptive cell transfer experiments in the FL-BM culture confirmed that the cDC2-gated MHCIIhi non-DCs were precursors to cDC2s, i.e., MHCIIhi pre-cDC2s. MHCIIhi pre-cDC2s also expressed the higher level of DC-specific transcription factor Zbtb46 as similarly as immature cDC2s. Besides, MHCIIhi pre-cDC2s were generated only from pre-cDCs and common DC progenitor (CDP) cells but not from monocytes and common monocyte progenitor (cMoP) cells, verifying that MHCIIhi pre-cDC2s are close lineage to cDCs. All in all, our study identified and characterized a new cDC precursor, exhibiting a CD11c+MHCIIhiCD115+CX3CR1+ phenotype, in FL-BM culture.
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Médula Ósea , Antígenos de Histocompatibilidad Clase II , Ratones , Animales , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Receptor 1 de Quimiocinas CX3C/metabolismo , Células de la Médula Ósea , Células Dendríticas , Diferenciación Celular , Proteínas Tirosina Quinasas Receptoras/metabolismoRESUMEN
BACKGROUND: Psoriasis is a chronically relapsing inflammatory skin disease primarily perpetuated by skin-resident IL-17-producing T (T17) cells. Pellino-1 (Peli1) belongs to a member of E3 ubiquitin ligase mediating immune receptor signaling cascades, including nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) pathway. OBJECTIVE: We explored the potential role of Peli1 in psoriatic inflammation in the context of skin-resident T17 cells. METHODS: We performed single-cell RNA sequencing of relapsing and resolved psoriatic lesions with analysis for validation data set of psoriasis. Mice with systemic and conditional depletion of Peli1 were generated to evaluate the role of Peli1 in imiquimod-induced psoriasiform dermatitis. Pharmacologic inhibition of Peli1 in human CD4+ T cells and ex vivo human skin cultures was also examined to evaluate its potential therapeutic implications. RESULTS: Single-cell RNA sequencing analysis revealed distinct T-cell subsets in relapsing psoriasis exhibiting highly enriched gene signatures for (1) tissue-resident T cells, (2) T17 cells, and (3) NF-κB signaling pathway including PELI1. Peli1-deficient mice were profoundly protected from psoriasiform dermatitis, with reduced IL-17A production and NF-κB activation in γδ T17 cells. Mice with conditional depletion of Peli1 treated with FTY720 revealed that Peli1 was intrinsically required for the skin-resident T17 cell immune responses. Notably, pharmacologic inhibition of Peli1 significantly ameliorated murine psoriasiform dermatitis and IL-17A production from the stimulated human CD4+ T cells and ex vivo skin explants modeling psoriasis. CONCLUSION: Targeting Peli1 would be a promising therapeutic strategy for psoriasis by limiting skin-resident T17 cell immune responses.
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Dermatitis , Psoriasis , Ratones , Humanos , Animales , Interleucina-17 , FN-kappa B/metabolismo , Piel , Modelos Animales de Enfermedad , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
BACKGROUND: Both human and mouse allergic contact dermatitis (ACD) frequently demonstrates a combined type 1 and type 2 immune response. However, the relative importance of type 2 immunity in this setting has been incompletely understood yet. OBJECTIVE: To explore an effector function of type 2 immunity in ACD with mixed type 1/type 2 immune response. METHODS: Gene expression characteristics of contact hypersensitivity (CHS) model was examined by quantitative polymerase chain reaction. Cytokine profile of T cells was analyzed by flow cytometry. The involvement of type 2 immunity was assessed by antibody-mediated cytokine neutralization and cell depletion. The role of specific subset of cutaneous dendritic cells was evaluated using diphtheria toxin-induced cell-depleting mouse strains. RESULTS: Oxazolone-induced CHS revealed a combination of type 1/type 2 gene expression. The severity of oxazolone-induced CHS was ameliorated by neutralization of IL-4 but not of IFN-γ, indicating that type 2 immunity plays a dominant effector function in this mixed type 1/type 2 model. Mechanistically, type 2 effector immunity was mounted by CD301b+Langeirn- dermal dendritic cells in part through thymic stromal lymphopoietin-interleukin 7 receptor alpha signaling-dependent manner. CONCLUSION: Our findings suggest the clinical rationale for targeting type 2 immunity as a relevant therapeutic strategy for the mixed immune phenotype of ACD.
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Células Dendríticas/inmunología , Dermatitis Alérgica por Contacto/genética , Dermatitis Alérgica por Contacto/inmunología , Animales , Antígenos de Superficie/metabolismo , Dermatitis Alérgica por Contacto/patología , Modelos Animales de Enfermedad , Inmunidad/genética , Inmunoglobulinas/metabolismo , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-17/genética , Interleucina-4/genética , Interleucina-4/inmunología , Lectinas Tipo C/metabolismo , Lectinas de Unión a Manosa/metabolismo , Ratones , Oxazolona , Receptores de Citocinas/metabolismo , Transducción de Señal , Piel/patología , Linfocitos T/metabolismo , TranscriptomaRESUMEN
The skin is the outermost barrier organ in the body, which contains several types of dendritic cells (DCs), a group of professional antigen-presenting cells. When the skin encounters invading pathogens, different cutaneous DCs initiate a distinct T cell immune response to protect the body. Among the invading pathogens, fungal infection specifically drives a protective interleukin-17-producing Th17 immune response. A protocol was developed to efficiently differentiate Th17 cells by intradermal Candida albicans infection to investigate a subset of cutaneous DCs responsible for inducing Th17 immunity. Flow cytometry and gene expression analyses revealed a prominent induction of Th17 immune response in skin-draining lymph nodes and infected skin. Using diphtheria toxin-induced DC subset-depleting mouse strains, CD301b+ dermal DCs were found to be responsible for mounting optimal Th17 differentiation in this model. Thus, this protocol provides a valuable method to study in vivo function of differential subsets of cutaneous DCs to determine Th17 immunity against deep skin fungal infection.
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Candida albicans , Células Th17 , Animales , Células Dendríticas , Células de Langerhans , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Langerhans cells (LCs) are dendritic cells that reside in the epidermis and local inflammation results in an increased differentiation of monocyte-derived LCs. Only few studies have investigated on the role of LCs in psoriasis-like dermatitis model, but the results are variable and the exact role of LCs in psoriasis model remains to be elucidated. OBJECTIVE: To explore the functional role of resident (rLCs) and monocyte-derived LCs (mLCs) in imiquimod (IMQ)-induced psoriasis-like inflammation using human Langerin-diphtheria toxin subunit A (huLang-DTA) mice. METHODS: 5% IMQ cream was topically applied on the skins. Clinical and histopathological features were evaluated. Psoriasis-related gene expression was analyzed by quantitative polymerase chain reaction. The production of psoriasis-related cytokines including IL-17A and IL-22 by T cells were assessed by flow cytometry from the lesional skins. RESULTS: huLang-DTA mice showed a common depletion of both rLCs and mLCs in the IMQ-treated skins. huLang-DTA mice had a reduced IMQ-induced psoriasis-like inflammation featuring erythema, scale, and thickness compared with wild-type mice. Psoriatic lesions from huLang-DTA mice had a decreased level of Il23a and accordingly demonstrated an attenuated cytokine production of IL-17A and IL-22 from γδlow T cells. mLCs revealed a significantly greater level of IL-23 expression compared to rLCs in response to topical IMQ treatment. CONCLUSION: Although both rLCs and mLCs are involved in the development of IMQ-induced psoriasis-like dermatitis, inflammation-induced mLCs present a superior capacity for producing IL-23 in this murine experimental model of psoriasis.
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Citocinas/metabolismo , Células de Langerhans/inmunología , Psoriasis/inmunología , Aminoquinolinas/inmunología , Animales , Antígenos CD/genética , Citocinas/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Células Epidérmicas , Epidermis/inmunología , Epidermis/metabolismo , Epidermis/patología , Femenino , Perfilación de la Expresión Génica , Humanos , Imiquimod , Células de Langerhans/metabolismo , Lectinas Tipo C/genética , Masculino , Lectinas de Unión a Manosa/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Monocitos/fisiología , Psoriasis/patologíaRESUMEN
Conventional dendritic cells (cDCs) are composed of heterogeneous subsets commonly arising from dendritic cell (DC)-committed progenitors. A population of CD301b-expressing DCs has recently been identified in non-lymphoid barrier tissues such as skin. However, whether CD301b+ DCs in the skin represent an ontogenetically unique subpopulation of migratory cDCs has not been fully addressed. Here, we demonstrated that CD301b+ dermal DCs were distinct subpopulation of FMS-like tyrosine kinase 3 ligand (FLT3L)-dependent CD11b+ cDC2 lineage, which required an additional GM-CSF cue for the adequate development. Although the majority of lymphoid-resident cDC2 lacked CD301b expression, dermal migratory cDC2 contained a substantial fraction of CD301b+ subset. Similar to CD301b- population, CD301b+ dermal DC development was closely regulated by FLT3 signaling, suggesting their common origin from FLT3L-responsive cDC progenitors. However, FLT3L-driven cDC progenitor culture was not sufficient, but additional GM-CSF treatment was required to produce CD301b+ cDC2. In vivo development of CD301b+ cDC2 was significantly augmented by exogenous GM-CSF, while the repopulation of CD301b+ dermal cDC2 was abrogated by GM-CSF neutralization. Functionally, CD301b+ cDC2 was capable of producing a high level of IL-23, and the depletion of CD301b+ cDC2 effectively prevented IL-17-mediated psoriasiform dermatitis. Therefore, our findings highlight the differentiation program of a distinct CD301b+ dermal cDC2 subset in the skin and its involvement in psoriatic inflammation.
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Células Dendríticas/inmunología , Dermis/patología , Inmunidad Celular , Interleucina-17/metabolismo , Lectinas Tipo C/inmunología , Psoriasis/inmunología , Animales , Células Cultivadas , Células Dendríticas/metabolismo , Células Dendríticas/patología , Dermis/inmunología , Dermis/metabolismo , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Psoriasis/metabolismo , Psoriasis/patología , Transducción de SeñalAsunto(s)
Citocinas/metabolismo , Proteína Antagonista del Receptor de Interleucina 1/metabolismo , Interleucinas/metabolismo , Psoriasis/metabolismo , Células Epidérmicas , Humanos , Proteína Antagonista del Receptor de Interleucina 1/sangre , Interleucinas/sangre , Queratinocitos/metabolismo , Leucocitos Mononucleares/metabolismo , Psoriasis/sangre , Células Th17/metabolismo , Regulación hacia ArribaRESUMEN
A Core Protection Calculator System (CPCS) was developed to initiate a Reactor Trip under the circumstance of certain transients by a Combustion Engineering Company. The major function of the Core Protection Calculator System is to generate contact outputs for the Departure from Nucleate Boiling Ratio (DNBR) Trip and a Local Power Density (LPD) Trip. But in a Core Protection Calculator System, a trip cause cannot be identified, thus only trip signals are transferred to the Plant Protection System (PPS) and only the trip status is displayed. It could take a considerable amount of time and effort for a plant operator to analyze the trip causes of a Core Protection Calculator System. So, a Cause Analysis System for a Core Protection Calculator System (CASCPCS) has been developed by using the rule-base deduction method to assist operators in a Nuclear Power Plant. CASCPCS consists of three major parts. Inference engine has a role of controlling the searching knowledge base, executing the rules and tracking the inference process by using the depth-first searching method. Knowledge base consists of four major parts: rules, data base constants, trip buffer variables and causes. And a user interface is implemented by using menu-driven and window display techniques. The advantage of CASCPCS is that it saves time and effort to diagnose the trip causes of a Core Protection Calculator System, it increases a plant's availability and reliability, and it makes it easy to manage CASCPCS because of using only a cursor control.