RESUMO
Squamous cell carcinoma (SCC) is the second most frequent skin cancer. The cellular origin of SCC remains controversial. Here, we used mouse genetics to determine the epidermal cell lineages at the origin of SCC. Using mice conditionally expressing a constitutively active KRas mutant (G12D) and an inducible CRE recombinase in different epidermal lineages, we activated Ras signaling in different cellular compartments of the skin epidermis and determined from which epidermal compartments Ras activation induces squamous tumor formation. Expression of mutant KRas in hair follicle bulge stem cells (SCs) and their immediate progeny (hair germ and outer root sheath), but not in their transient amplifying matrix cells, led to benign squamous skin tumor (papilloma). Expression of KRas(G12D) in interfollicular epidermis also led to papilloma formation, demonstrating that squamous tumor initiation is not restricted to the hair follicle lineages. Whereas no malignant tumor was observed after KRas(G12D) expression alone, expression of KRas(G12D) combined with the loss of p53 induced invasive SCC. Our studies demonstrate that different epidermal lineages including bulge SC are competent to initiate papilloma formation and that multiple genetic hits in the context of oncogenic KRas are required for the development of invasive SCC.
Assuntos
Carcinoma de Células Escamosas/fisiopatologia , Linhagem da Célula/fisiologia , Células Epidérmicas , Neoplasias Cutâneas/fisiopatologia , Animais , Bromodesoxiuridina , Carcinoma de Células Escamosas/genética , Primers do DNA/genética , Citometria de Fluxo , Folículo Piloso/citologia , Integrases/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/genética , Células-Tronco/metabolismoRESUMO
Th17-mediated immune responses have been recently identified as novel pathogenic mechanisms in a variety of conditions; however, their importance in allograft rejection processes is still debated. In this paper, we searched for MHC or minor Ag disparate models of skin graft rejection in which Th17 immune responses might be involved. We found that T cell-derived IL-17 is critical for spontaneous rejection of minor but not major Ag-mismatched skin grafts. IL-17 neutralization was associated with a lack of neutrophil infiltration and neutrophil depletion delayed rejection, suggesting neutrophils as an effector mechanism downstream of Th17 cells. Regulatory T cells (Tregs) appeared to be involved in Th17 reactivity. We found that in vivo Treg depletion prevented IL-17 production by recipient T cells. An adoptive cotransfer of Tregs with naive monospecific antidonor T cells in lymphopenic hosts biased the immune response toward Th17. Finally, we observed that IL-6 was central for balancing Tregs and Th17 cells as demonstrated by the prevention of Th17 differentiation, the enhanced Treg/Th17 ratio, and a net impact of rejection blockade in the absence of IL-6. In conclusion, the ability of Tregs to promote the Th17/neutrophil-mediated pathway of rejection that we have described should be considered as a potential drawback of Treg-based cell therapy.
Assuntos
Rejeição de Enxerto/genética , Interleucina-17/fisiologia , Antígenos de Histocompatibilidade Menor/genética , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Feminino , Técnicas de Introdução de Genes , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Interleucina-17/deficiência , Interleucina-17/genética , Teste de Cultura Mista de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Transplante de Pele/imunologia , Transplante de Pele/patologia , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Reguladores/metabolismoRESUMO
In bone marrow-transplanted patients, chronic graft-versus-host disease is a complication that results from the persistent stimulation of recipient minor histocompatibility Ag (mHA)-specific T cells contained within the graft. In this study, we developed a mouse model where persistent stimulation of donor T cells by recipient's mHA led to multiorgan T cell infiltration. Exposure to systemic mHA, however, deeply modified T cell function and chronically stimulated T cells developed a long-lasting state of unresponsiveness, or immune adaptation, characterized by their inability to mediate organ immune damages in vivo. However, analysis of the gene expression profile of adapted CD4+ T cells revealed the specific coexpression of genes known to promote differentiation and function of Th1 effector cells as well as genes coding for proteins that control T cell activity, such as cell surface-negative costimulatory molecules and regulatory cytokines. Strikingly, blockade of negative costimulation abolished T cell adaptation and stimulated strong IFN-gamma production and severe multiorgan wasting disease. Negative costimulation was also shown to control lethal LPS-induced toxic shock in mice with adapted T cells, as well as the capacity of adapted T cells to reject skin graft. Our results demonstrate that negative costimulation is the molecular mechanism used by CD4+ T cells to adapt their activity in response to persistent antigenic stimulation. The effector function of CD4+ T cells that have adapted to chronic Ag presentation can be activated by stimuli strong enough to overcome regulatory signals delivered to the T cells by negative costimulation.
Assuntos
Apresentação de Antígeno/imunologia , Linfócitos T CD4-Positivos/imunologia , Tolerância Imunológica , Sequência de Aminoácidos , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/transplante , Células Cultivadas , Feminino , Antígeno H-Y/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Antígenos de Histocompatibilidade Menor/imunologia , Dados de Sequência Molecular , Transplante de Pele/imunologiaRESUMO
Tumor-associated macrophages (TAMs) contribute to the maintenance of a strong immunosuppressive environment, supporting tumor progression and resistance to treatment. To date, the mechanisms that drive acquisition of these immunosuppressive features are still poorly defined. Heme oxygenase-1 (HO-1) is the rate-limiting enzyme that catabolizes free heme. It displays important cytoprotective, antiinflammatory, and antioxidant properties. A growing body of evidence suggests that HO-1 may also promote tumor development. Herein, we show that HO-1 is highly expressed in monocytic cells in the tumor microenvironment (TME) once they differentiate into TAMs. Deletion of HO-1 in the myeloid compartment enhances the beneficial effects of a therapeutic antitumor vaccine by restoring CD8+ T cell proliferation and cytotoxicity. We further show that induction of HO-1 plays a major role in monocyte education by tumor cells by modulating their transcriptional and epigenetic programs. These results identify HO-1 as a valuable therapeutic target to reprogram the TME and synergize with current cancer therapies to facilitate antitumor response.
Assuntos
Heme Oxigenase-1/imunologia , Tolerância Imunológica , Proteínas de Membrana/imunologia , Neoplasias/imunologia , Microambiente Tumoral/imunologia , Macrófagos Associados a Tumor/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Heme Oxigenase-1/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Neoplasias/genética , Microambiente Tumoral/genética , Macrófagos Associados a Tumor/patologiaRESUMO
Graft-versus-host disease (GVHD) remains a major clinical drawback of allogeneic hematopoietic stem cell transplantation (HSCT). Here, we investigated how the stress responsive heme catabolizing enzyme heme oxygenase-1 (HO-1, encoded by HMOX1) regulates GVHD in response to allogeneic hematopoietic stem cell transplantation in mice and humans. We found that deletion of the Hmox1 allele, specifically in the myeloid compartment of mouse donor bone marrow, promotes the development of aggressive GVHD after allogeneic transplantation. The mechanism driving GVHD in mice transplanted with allogeneic bone marrow lacking HO-1 expression in the myeloid compartment involves enhanced T cell alloreactivity. The clinical relevance of these observations was validated in two independent cohorts of HSCT patients. Individuals transplanted with hematopoietic stem cells from donors carrying a long homozygous (GT)n repeat polymorphism (L/L) in the HMOX1 promoter, which is associated with lower HO-1 expression, were at higher risk of developing severe acute GVHD as compared to donors carrying a short (GT)n repeat (S/L or S/S) polymorphism associated with higher HO-1 expression. In this study, we showed the unique importance of donor-derived myeloid HO-1 in the prevention of lethal experimental GVHD and we corroborated this observation by demonstrating the association between human HMOX1 (GT)n microsatellite polymorphisms and the incidence of severe acute GVHD in two independent HSCT patient cohorts. Donor-derived myeloid HO-1 constitutes a potential therapeutic target for HSCT patients and large-scale prospective studies in HSCT patients are necessary to validate the HO-1 L/L genotype as an independent risk factor for developing severe acute GVHD.
Assuntos
Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Heme Oxigenase-1/metabolismo , Proteínas de Membrana/metabolismo , Células Supressoras Mieloides/transplante , Adulto , Animais , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Doença Enxerto-Hospedeiro/enzimologia , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/imunologia , Heme Oxigenase-1/genética , Homozigoto , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Repetições de Microssatélites , Pessoa de Meia-Idade , Células Supressoras Mieloides/enzimologia , Fenótipo , Polimorfismo Genético , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Índice de Gravidade de DoençaRESUMO
We report a new case of hypobetalipoproteinemia in a 44-year-old man of Peruvian origin exhibiting a heterozygous PCSK9 missense mutation (c.946 G>T, p. Gly316Cys). In vitro functional studies demonstrated that this mutation leads to a loss of function of PCSK9 on low-density lipoprotein receptor degradation. This patient exhibited liver steatosis; he was neither diabetic, nor obese or alcoholic, but is a carrier of 2 polymorphisms, p.Ile148Met (rs738409) and p.Glu167Lys (rs58542926) on PNPLA3 and TM6SF2 gene, respectively, previously shown to be associated with nonalcoholic steatosis and fibrosis evolution. These data suggested that if a resistance to hepatic steatosis mediated by the PCSK9 deficiency exists, as demonstrated in mice, it is not sufficient to prevent hepatic fatty accumulation in the case of genetic factors predisposing to nonalcoholic fatty liver disease.
Assuntos
Colesterol/metabolismo , Predisposição Genética para Doença/genética , Mutação com Perda de Função , Hepatopatia Gordurosa não Alcoólica/enzimologia , Hepatopatia Gordurosa não Alcoólica/genética , Polimorfismo Genético , Pró-Proteína Convertase 9/genética , Adulto , Humanos , Masculino , Hepatopatia Gordurosa não Alcoólica/metabolismoAssuntos
Imidazóis , Tetrazóis , Bélgica/epidemiologia , Diarreia , Humanos , Imidazóis/efeitos adversos , Tetrazóis/efeitos adversosRESUMO
Several evidences suggest that regulatory T cells (Treg) promote Th17 differentiation. Based on this hypothesis, we tested the effect of IL-17A neutralization in a model of skin transplantation in which long-term graft survival depends on a strong in vivo Treg expansion induced by transient exogenous IL-2 administration. As expected, IL-2 supplementation prevented rejection of MHC class II disparate skin allografts but, surprisingly, not in IL-17A-deficient recipients. We attested that IL-17A was not required for IL-2-mediated Treg expansion, intragraft recruitment or suppressive capacities. Instead, IL-17A prevented allograft rejection by inhibiting Th1 alloreactivity independently of Tregs. Indeed, T-bet expression of naive alloreactive CD4+ T cells and the subsequent Th1 immune response was significantly enhanced in IL-17A deficient mice. Our results illustrate for the first time a protective role of IL-17A in CD4+-mediated allograft rejection process.
Assuntos
Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/imunologia , Interleucina-17/imunologia , Interleucina-2/farmacologia , Transplante de Pele , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Animais , Comunicação Celular , Proliferação de Células , Expressão Gênica , Rejeição de Enxerto/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Interleucina-17/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Proteínas com Domínio T/genética , Proteínas com Domínio T/imunologia , Linfócitos T Reguladores/metabolismo , Células Th1/metabolismo , Células Th17/imunologia , Células Th17/metabolismoRESUMO
Primary graft dysfunction (PGD) and bronchiolitis obliterans (BO) are the leading causes of morbidity and mortality after lung transplantation. Reports from clinical and rodent models suggest the implication of IL-17A in either PGD or BO. We took advantage of the heterotopic trachea transplantation model in mice to study the direct role of IL-17A in post-transplant airway lesions. Across full MHC barrier, early lesions were controlled in IL-17A(-/-) or anti-IL17 treated recipients. In contrast, IL-17A deficiency did not prevent subsequent obliterative airway disease (OAD). Interestingly, this early protection occurred also in syngeneic grafts and was accompanied by a decrease in cellular stress, as attested by lower HSP70 mRNA levels, suggesting the involvement of IL-17A in ischemia-reperfusion injury (IRI). Furthermore, persistence of multipotent CK14(+) epithelial stem cells underlined allograft protection afforded by IL-17A deficiency or neutralisation. Recipient-derived γδ(+) and CD4(+) T cells were the major source of IL-17A. However, lesions still occurred in the absence of each subset, suggesting a high redundancy between the innate and adaptive IL-17A producing cells. Notably, a double depletion significantly diminished lesions. In conclusion, this work implicated IL-17A as mediator of early post-transplant airway lesions and could be considered as a potential therapeutic target in clinical transplantation.
Assuntos
Interleucina-17/metabolismo , Traqueia/metabolismo , Traqueia/patologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/metabolismo , Rejeição de Enxerto/patologia , Interleucina-17/deficiência , Interleucina-17/genética , Transplante de Pulmão/efeitos adversos , Camundongos , Camundongos Knockout , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Traumatismo por Reperfusão/complicações , Traqueia/transplante , Transplante Heterotópico , Transplante HomólogoRESUMO
Activation of innate immunity through Toll-like receptors (TLR) can abrogate transplantation tolerance by revealing hidden T cell alloreactivity. Separately, the cholinergic anti-inflammatory pathway has the capacity to dampen macrophage activation and cytokine release during endotoxemia and ischemia reperfusion injury. However, the relevance of the α7 nicotinic acetylcholine receptor (α7nAChR)-dependent anti-inflammatory pathway in the process of allograft rejection or maintenance of tolerance remains unknown. The aim of our study is to investigate whether the cholinergic pathway could impact T cell alloreactivity and transplant outcome in mice. For this purpose, we performed minor-mismatched skin allografts using donor/recipient combinations genetically deficient for the α7nAChR. Minor-mismatched skin grafts were not rejected unless the mice were housed in an environment with endogenous pathogen exposure or the graft was treated with direct application of imiquimod (a TLR7 ligand). The α7nAChR-deficient recipient mice showed accelerated rejection compared to wild type recipient mice under these conditions of TLR activation. The accelerated rejection was associated with enhanced IL-17 and IFN-γ production by alloreactive T cells. An α7nAChR-deficiency in the donor tissue facilitated allograft rejection but not in recipient mice. In addition, adoptive T cell transfer experiments in skin-grafted lymphopenic animals revealed a direct regulatory role for the α7nAChR on T cells. Taken together, our data demonstrate that the cholinergic pathway regulates alloreactivity and transplantation tolerance at multiple levels. One implication suggested by our work is that, in an organ transplant setting, deliberate α7nAChR stimulation of brain dead donors might be a valuable approach for preventing donor tissue inflammation prior to transplant.
Assuntos
Aloenxertos , Rejeição de Enxerto , Inflamação/prevenção & controle , Transplante de Pele , Receptores Toll-Like/fisiologia , Receptor Nicotínico de Acetilcolina alfa7/fisiologia , Transferência Adotiva , Animais , Sequência de Bases , Células Cultivadas , Primers do DNA , Feminino , Citometria de Fluxo , Inflamação/metabolismo , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/transplanteRESUMO
Interleukin (IL) 17 is a proinflammatory cytokine already known to play a defense role against microbes and a pathogenic role in a number of autoimmune diseases. Although IL-17 can be produced by a variety of cells including neutrophils, CD8+, NK, and gamma-delta T cells, the concept of IL-17-secreting CD4+ T helper cells (Th17), distinct from Th1 and Th2, recently emerged. Herein, we discuss arguments in favor of a Th17-mediated alternative pathway of allograft rejection based on clinical and experimental observations drawn from the literature. We also discuss the complex interplays among regulatory T cells and Th17 cells in the allogeneic context.