RESUMO
OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant desmoplastic stroma composed of cancer-associated fibroblasts (CAF) and interspersed immune cells. A non-canonical CD8+ T-cell subpopulation producing IL-17A (Tc17) promotes autoimmunity and has been identified in tumours. Here, we evaluated the Tc17 role in PDAC. DESIGN: Infiltration of Tc17 cells in PDAC tissue was correlated with patient overall survival and tumour stage. Wild-type (WT) or Il17ra-/- quiescent pancreatic stellate cells (qPSC) were exposed to conditional media obtained from Tc17 cells (Tc17-CM); moreover, co-culture of Tc17-CM-induced inflammatory (i)CAF (Tc17-iCAF) with tumour cells was performed. IL-17A/F-, IL-17RA-, RAG1-deficient and Foxn1nu/nu mice were used to study the Tc17 role in subcutaneous and orthotopic PDAC mouse models. RESULTS: Increased abundance of Tc17 cells highly correlated with reduced survival and advanced tumour stage in PDAC. Tc17-CM induced iCAF differentiation as assessed by the expression of iCAF-associated genes via synergism of IL-17A and TNF. Accordingly, IL-17RA controlled the responsiveness of qPSC to Tc17-CM. Pancreatic tumour cells co-cultured with Tc17-iCAF displayed enhanced proliferation and increased expression of genes implicated in proliferation, metabolism and protection from apoptosis. Tc17-iCAF accelerated growth of mouse and human tumours in Rag1-/- and Foxn1nu/nu mice, respectively. Finally, Il17ra-expressed by fibroblasts was required for Tc17-driven tumour growth in vivo. CONCLUSIONS: We identified Tc17 as a novel protumourigenic CD8+ T-cell subtype in PDAC, which accelerated tumour growth via IL-17RA-dependent stroma modification. We described a crosstalk between three cell types, Tc17, fibroblasts and tumour cells, promoting PDAC progression, which resulted in poor prognosis for patients.
Assuntos
Fibroblastos Associados a Câncer , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Linfócitos T CD8-Positivos , Fibroblastos Associados a Câncer/metabolismo , Interleucina-17/metabolismo , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/patologia , Proteínas de Homeodomínio , Neoplasias PancreáticasRESUMO
Keratinocytes (KCs) form the outer epithelial barrier of the body, protecting against invading pathogens. Mice lacking the IL-17RA or both IL-17A and IL-17F develop spontaneous Staphylococcusaureus skin infections. We found a marked expansion of T17 cells, comprised of RORγt-expressing γδ T cells and T helper 17 cells in the skin-draining lymph nodes of these mice. Contradictory to previous suggestions, this expansion was not a result of a direct negative feedback loop because we found no expansion of T17 cells in mice lacking IL-17 signaling specifically in T cells. Instead, we found that the T17 expansion depended on the microbiota and was observed only when KCs were deficient for IL-17RA signaling. Indeed, mice that lack IL-17RA only in KCs showed an increased susceptibility to experimental epicutaneous infection with S. aureus together with an accumulation of IL-17A-producing γδ T cells. We conclude that deficiency of IL-17RA on KCs leads to microbiota dysbiosis in the skin, which triggers the expansion of IL-17A-producing T cells. Our data show that KCs are the primary target cells of IL-17A and IL-17F, coordinating the defense against microbial invaders in the skin.
Assuntos
Interleucina-17 , Staphylococcus aureus , Camundongos , Animais , Camundongos Knockout , Pele , Queratinócitos , Camundongos Endogâmicos C57BLRESUMO
The proinflammatory cytokine interleukin 1 (IL-1) is crucially involved in the pathogenesis of multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Herein, we studied the role of IL-1 signaling in blood-brain barrier (BBB) endothelial cells (ECs), astrocytes and microglia for EAE development, using mice with the conditional deletion of its signaling receptor IL-1R1. We found that IL-1 signaling in microglia and astrocytes is redundant for the development of EAE, whereas the IL-1R1 deletion in BBB-ECs markedly ameliorated disease severity. IL-1 signaling in BBB-ECs upregulated the expression of the adhesion molecules Vcam-1, Icam-1 and the chemokine receptor Darc, all of which have been previously shown to promote CNS-specific inflammation. In contrast, IL-1R1 signaling suppressed the expression of the stress-responsive heme catabolizing enzyme heme oxygenase-1 (HO-1) in BBB-ECs, promoting disease progression via a mechanism associated with deregulated expression of the IL-1-responsive genes Vcam1, Icam1 and Ackr1 (Darc). Mechanistically, our data emphasize a functional crosstalk of BBB-EC IL-1 signaling and HO-1, controlling the transcription of downstream proinflammatory genes promoting the pathogenesis of autoimmune neuroinflammation.
Assuntos
Barreira Hematoencefálica/enzimologia , Encefalomielite Autoimune Experimental/imunologia , Células Endoteliais/enzimologia , Heme Oxigenase-1/metabolismo , Inflamação/imunologia , Interleucina-1/imunologia , Animais , Barreira Hematoencefálica/imunologia , Encefalomielite Autoimune Experimental/enzimologia , Regulação da Expressão Gênica/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/imunologiaRESUMO
IL-17-producing CD8+ (Tc17) cells are enriched in active lesions of patients with multiple sclerosis (MS), suggesting a role in the pathogenesis of autoimmunity. Here we show that amelioration of MS by dimethyl fumarate (DMF), a mechanistically elusive drug, associates with suppression of Tc17 cells. DMF treatment results in reduced frequency of Tc17, contrary to Th17 cells, and in a decreased ratio of the regulators RORC-to-TBX21, along with a shift towards cytotoxic T lymphocyte gene expression signature in CD8+ T cells from MS patients. Mechanistically, DMF potentiates the PI3K-AKT-FOXO1-T-BET pathway, thereby limiting IL-17 and RORγt expression as well as STAT5-signaling in a glutathione-dependent manner. This results in chromatin remodeling at the Il17 locus. Consequently, T-BET-deficiency in mice or inhibition of PI3K-AKT, STAT5 or reactive oxygen species prevents DMF-mediated Tc17 suppression. Overall, our data disclose a DMF-AKT-T-BET driven immune modulation and suggest putative therapy targets in MS and beyond.
Assuntos
Linfócitos T CD8-Positivos/efeitos dos fármacos , Fumarato de Dimetilo/farmacologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Esclerose Múltipla/tratamento farmacológico , Adolescente , Adulto , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Fumarato de Dimetilo/uso terapêutico , Encefalomielite Autoimune Experimental/sangue , Encefalomielite Autoimune Experimental/imunologia , Feminino , Humanos , Imunossupressores , Interleucina-17/imunologia , Interleucina-17/metabolismo , Estudos Longitudinais , Masculino , Camundongos , Pessoa de Meia-Idade , Esclerose Múltipla/sangue , Esclerose Múltipla/imunologia , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Resultado do Tratamento , Adulto JovemRESUMO
Dimethyl fumarate (DMF) is widely used to treat the human autoimmune diseases multiple sclerosis (MS) and psoriasis. DMF causes short-term oxidative stress and activates the antioxidant response via the transcription factor Nrf2 but its immunosuppressive effect is not well understood. Immune cell activation depends on calcium signaling which itself is influenced by the cellular redox state. We therefore measured calcium, reactive oxygen species levels and glutathione content in lymphocytes from immunized mice before onset of experimental autoimmune encephalomyelitis, in peripheral blood mononuclear cells from MS patients treated with DMF, and in mouse splenocytes treated ex vivo with DMF. This demonstrated altered redox states and increased lymphocytic calcium levels in all model systems. DMF caused an immediate influx of calcium from the extracellular space, long-term increased cytosolic calcium levels and reduced calcium stored in intracellular stores. The DMF-elicited current had the electrophysiological characteristics of a transient receptor potential channel and the intracellular calcium levels were normalized by antagonists of TRPA1. Interestingly, the sarco/endoplasmic reticulum Ca2+-ATPase SERCA2b was downregulated but more active due to glutathionylation of the redox-sensitive cysteine 674. DMF therefore causes pleiotropic changes in cellular calcium homeostasis which are likely caused by redox-sensitive post-translational modifications. These changes probably contribute to its immunosuppressive effects.
Assuntos
Fumarato de Dimetilo/farmacologia , Esclerose Múltipla/tratamento farmacológico , Psoríase/tratamento farmacológico , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Canal de Cátion TRPA1/genética , Animais , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa/metabolismo , Humanos , Linfócitos/efeitos dos fármacos , Camundongos , Esclerose Múltipla/genética , Esclerose Múltipla/patologia , Oxirredução/efeitos dos fármacos , Psoríase/genética , Psoríase/patologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Recently our groups discovered lugdunin, a new cyclic peptide antibiotic that inhibits Staphylococcus aureus epithelial colonization in humans and rodents. In this work, we analyzed its immuno-modulatory and antimicrobial potential as a single agent or in combination with other microbiota- or host-derived factors. We show that pretreatment of primary human keratinocytes or mouse skin with lugdunin in combination with microbiota-derived factors results in a significant reduction of S. aureus colonization. Moreover, lugdunin increases expression and release of LL-37 and CXCL8/MIP-2 in human keratinocytes and mouse skin, and results in the recruitment of monocytes and neutrophils in vivo, both by a TLR/MyD88-dependent mechanism. Interestingly, S. aureus elimination by lugdunin is additionally achieved by synergistic antimicrobial activity with LL-37 and dermcidin-derived peptides. In summary, our results indicate that lugdunin provides multi-level protection against S. aureus and may thus become a promising treatment option for S. aureus skin infections in the future.
Assuntos
Antibacterianos/farmacologia , Imunidade Inata/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Tiazolidinas/farmacologia , Animais , Antibacterianos/uso terapêutico , Peptídeos Catiônicos Antimicrobianos/imunologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/imunologia , Queratinócitos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microbiota/efeitos dos fármacos , Microbiota/imunologia , Peptídeos/imunologia , Peptídeos Cíclicos/uso terapêutico , Cultura Primária de Células , Pele/efeitos dos fármacos , Pele/imunologia , Pele/microbiologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Tiazolidinas/uso terapêutico , CatelicidinasRESUMO
The pathology of psoriasis strongly depends on IL-17A. Monoclonal antibodies blocking either the cytokine or its receptor are among the most efficient treatments for psoriatic patients. Keratinocytes can be activated upon exposure to IL-17A and tumor necrosis factor-α and secrete secondary cytokines and chemokines in the inflamed skin. In psoriasis and its imiquimod-induced mouse model, a strong skin infiltration of neutrophils and inflammatory monocytes can be observed. However, to date, it is not clear how exactly those cellular populations are attracted to the skin and how they contribute to the pathogenesis of the disease. To define the crucial cell type responding to IL-17 and initiating the downstream pathology in psoriasis-like dermatitis, we used mice specifically lacking the IL-17 receptor (IL-17RA) in different cell types. Deletion of IL-17RA in T cells or myeloid had no impact on disease development. Only deletion of this receptor in keratinocytes reflected the full-body deletion of IL-17RA, resulting in strongly reduced dermatitis development. Imiquimod treatment of those IL-17 signaling-deficient mice maintained high monocytic infiltration but failed to attract neutrophils into the skin. We conclude that keratinocytes are a critical cellular target for IL-17A-mediated neutrophil attraction and psoriasis development.
Assuntos
Adjuvantes Imunológicos/farmacologia , Imiquimode/farmacologia , Interleucina-17/genética , Psoríase/genética , Transdução de Sinais/efeitos dos fármacos , Animais , Biópsia por Agulha , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Imuno-Histoquímica , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Psoríase/induzido quimicamente , Psoríase/patologia , Distribuição Aleatória , Reação em Cadeia da Polimerase em Tempo Real/métodos , Transdução de Sinais/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
IL-17A and IL-17F share the highest sequence homology of the IL-17 family and signal via the same IL-17RA/RC receptor heterodimer. To better explore the expression of these two cytokines, we used a double reporter mouse strain (IL-17DR mice), where IL-17A expressing cells are marked by enhanced green fluorescent protein (eGFP) while red fluorescence protein (RFP) reports the expression of IL-17F. In steady state, we found that Th17 and γδ T cells only expressed IL-17A, while IL-17F expression was restricted to CD8 T cells (Tc17) and innate lymphoid cells (ILC type 3) of the gut. In experimental autoimmune encephalomyelitis, the vast majority of CNS-infiltrating Th17 cells expressed IL-17A but not IL-17F. In contrast, anti-CD3-induced, TGF-ß-driven Th17 cells in the gut expressed both of these IL-17 cytokines. In line with this, in vitro differentiation of Th17 cells in the presence of IL-1ß led primarily to IL-17A expressing T cells, while TGF-ß induced IL-17F co-expressing Th17 cells. Our results suggest that expression of IL-17F is associated with non-pathogenic T cells, pointing to a differential function of IL-17A versus IL-17F. KEY MESSAGES: Naïve mice: CD4+ T cells and γδ T cells express IL-17A, and Tc17 cells express IL-17F. Gut ILC3 show differential expression of IL17A and F. Th17 differentiation with TGF-ß1 induces IL-17A and F, whereas IL-1ß induced cells expressing IL-17A. Th17 cells in EAE in CNS express IL-17A only. Gut Th17 cells induced by anti-CD3 express IL-17A and F together as skin γδ T cells of IMQ-treated mice.
Assuntos
Expressão Gênica , Interleucina-17/genética , Células Th17/metabolismo , Animais , Biomarcadores , Diferenciação Celular/imunologia , Suscetibilidade a Doenças , Encefalomielite Autoimune Experimental , Imunofenotipagem , Interleucina-17/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Camundongos Transgênicos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Th17/citologia , Células Th17/imunologiaRESUMO
Cofilin is an essential actin remodeling protein promoting depolymerization and severing of actin filaments. To address the relevance of cofilin for the development and function of T cells in vivo, we generated knock-in mice in which T-cell-specific nonfunctional (nf) cofilin was expressed instead of wild-type (WT) cofilin. Nf cofilin mice lacked peripheral αß T cells and showed a severe thymus atrophy. This was caused by an early developmental arrest of thymocytes at the double negative (DN) stage. Importantly, even though DN thymocytes expressed the TCRß chain intracellularly, they completely lacked TCRß surface expression. In contrast, nf cofilin mice possessed normal numbers of γδ T cells. Their functionality was confirmed in the γδ T-cell-driven, imiquimod (IMQ)-induced, psoriasis-like murine model. Overall, this study not only highlights the importance of cofilin for early αß T-cell development but also shows for the first time that an actin-binding protein is differentially involved in αß versus γδ T-cell development.
Assuntos
Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Timo/metabolismo , Fatores de Despolimerização de Actina/química , Animais , Movimento Celular , Técnicas de Introdução de Genes , Humanos , Células Jurkat , Camundongos , Mutação/genética , Prolina/metabolismo , Subpopulações de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Timócitos/metabolismoRESUMO
IL-22 is a potent pro-inflammatory cytokine upregulated in psoriasis and in other inflammatory diseases. The function of IL-22 is regulated by the soluble scavenging receptor, IL-22 binding protein (IL-22BP or IL-22RA2). However, the role and regulation of IL-22BP itself in the pathogenesis of inflammatory disease remain unclear. We used the TLR7 agonist Imiquimod (IMQ) to induce a psoriasis-like skin disease in mice and found a strong downregulation of IL-22BP in the affected skin as well as in the lymph nodes of animals treated with IMQ. We also analysed psoriatic skin of patients and compared this to skin of healthy donors. Interestingly, IL-22BP expression was similarly downregulated in skin biopsies of psoriasis patients compared to the skin of healthy donors. Since IL-22BP is expressed foremost in dendritic cells, we characterized its expression in monocyte-derived dendritic cells (MoDC) during maturation. In this way, we found Prostaglandin E2 (PGE2) to be a potent suppressor of IL-22BP expression in vitro. We conclude that regulation of IL-22BP by inflammatory mediators is an important step for the progression of inflammation in the skin and possibly also in other autoimmune diseases.
Assuntos
Psoríase/imunologia , Receptores de Interleucina/imunologia , Animais , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/patologia , Dinoprostona/análise , Dinoprostona/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Psoríase/patologia , Receptores de Interleucina/análise , Pele/imunologia , Pele/patologiaRESUMO
GPCR expression was intensively studied in bulk cDNA of leukocyte populations, but limited data are available with respect to expression in individual cells. Here, we show a microfluidic-based single-cell GPCR expression analysis in primary T cells, myeloid cells, and endothelial cells under naive conditions and during experimental autoimmune encephalomyelitis, the mouse model of multiple sclerosis. We found that neuroinflammation induces characteristic changes in GPCR heterogeneity and patterning, and we identify various functionally relevant subgroups with specific GPCR profiles among spinal cord-infiltrating CD4 T cells, macrophages, microglia, or endothelial cells. Using GPCRs CXCR4, S1P1, and LPHN2 as examples, we show how this information can be used to develop new strategies for the functional modulation of Th17 cells and activated endothelial cells. Taken together, single-cell GPCR expression analysis identifies functionally relevant subpopulations with specific GPCR repertoires and provides a basis for the development of new therapeutic strategies in immune disorders.
RESUMO
The cytokine IL-17 is now a target for an array of therapeutic monoclonal antibodies supposed to treat a variety of inflammatory diseases. The forerunner Secukinumab, an IL-17A neutralizing antibody, is meanwhile approved as first-line treatments for moderate-to-severe plaque psoriasis, and as second-line treatment for psoriatic arthritis and ankylosing spondylitis. Ixekizumab and Brodalumab, both also targeting the IL-17 pathway, were also recently approved by the FDA for plaque psoriasis. Using mice overexpressing IL-17A in a tissue of choice, we showed that the ectopic expression of this cytokine in keratinocytes resulted in a spontaneous and very strong form of psoriasis-like dermatitis. Interestingly, this model showed some typical comorbidities found in humans with psoriasis. In this review, we will discuss why IL-17 is a good target especially in psoriasis and what we learned from mouse models about its functions in pathological situations.
Assuntos
Dermatite/imunologia , Interleucina-17/antagonistas & inibidores , Interleucina-17/imunologia , Psoríase/tratamento farmacológico , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Dermatite/patologia , Modelos Animais de Doenças , Humanos , Interleucina-17/genética , Interleucina-17/metabolismo , Camundongos , Camundongos Transgênicos , Terapia de Alvo Molecular/métodos , Psoríase/imunologia , Transdução de Sinais/imunologia , Pele/imunologia , Pele/patologia , Espondilite Anquilosante/tratamento farmacológicoRESUMO
Arrival of encephalitogenic T cells at inflammatory foci represents a critical step in development of experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis. EBI2 and its ligand, 7α,25-OHC, direct immune cell localization in secondary lymphoid organs. CH25H and CYP7B1 hydroxylate cholesterol to 7α,25-OHC. During EAE, we found increased expression of CH25H by microglia and CYP7B1 by CNS-infiltrating immune cells elevating the ligand concentration in the CNS. Two critical pro-inflammatory cytokines, interleukin-23 (IL-23) and interleukin-1 beta (IL-1ß), maintained expression of EBI2 in differentiating Th17 cells. In line with this, EBI2 enhanced early migration of encephalitogenic T cells into the CNS in a transfer EAE model. Nonetheless, EBI2 was dispensable in active EAE. Human Th17 cells do also express EBI2, and EBI2 expressing cells are abundant within multiple sclerosis (MS) white matter lesions. These findings implicate EBI2 as a mediator of CNS autoimmunity and describe mechanistically its contribution to the migration of autoreactive T cells into inflamed organs.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/fisiologia , Movimento Celular/fisiologia , Sistema Nervoso Central/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Esclerose Múltipla/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Autoimunidade/fisiologia , Sistema Nervoso Central/fisiologia , Família 7 do Citocromo P450/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/patologia , Feminino , Interleucina-1beta/metabolismo , Interleucina-23/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Esteroide Hidroxilases/metabolismo , Células Th17/metabolismo , Células Th17/fisiologiaRESUMO
Staining of transcription factors (TFs) together with retention of fluorescent reporter proteins is hindered by loss of fluorescence using current available methods. In this study, it is shown that current TF staining protocols do not destroy fluorescent proteins (FPs) but rather that fixation is not sufficient to retain FPs in the cytosol of the permeabilized cells. In this article, a simple and reliable protocol is elaborated, which allows efficient TF and cytokine staining while retaining FPs inside fixed cells.