RESUMEN
Coeliac disease is a complex, polygenic inflammatory enteropathy caused by exposure to dietary gluten that occurs in a subset of genetically susceptible individuals who express either the HLA-DQ8 or HLA-DQ2 haplotypes1,2. The need to develop non-dietary treatments is now widely recognized3, but no pathophysiologically relevant gluten- and HLA-dependent preclinical model exists. Furthermore, although studies in humans have led to major advances in our understanding of the pathogenesis of coeliac disease4, the respective roles of disease-predisposing HLA molecules, and of adaptive and innate immunity in the development of tissue damage, have not been directly demonstrated. Here we describe a mouse model that reproduces the overexpression of interleukin-15 (IL-15) in the gut epithelium and lamina propria that is characteristic of active coeliac disease, expresses the predisposing HLA-DQ8 molecule, and develops villous atrophy after ingestion of gluten. Overexpression of IL-15 in both the epithelium and the lamina propria is required for the development of villous atrophy, which demonstrates the location-dependent central role of IL-15 in the pathogenesis of coeliac disease. In addition, CD4+ T cells and HLA-DQ8 have a crucial role in the licensing of cytotoxic T cells to mediate intestinal epithelial cell lysis. We also demonstrate a role for the cytokine interferon-γ (IFNγ) and the enzyme transglutaminase 2 (TG2) in tissue destruction. By reflecting the complex interaction between gluten, genetics and IL-15-driven tissue inflammation, this mouse model provides the opportunity to both increase our understanding of coeliac disease, and develop new therapeutic strategies.
Asunto(s)
Enfermedad Celíaca/inmunología , Enfermedad Celíaca/patología , Glútenes/inmunología , Antígenos HLA-DQ/inmunología , Interleucina-15/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Femenino , Antígenos HLA-DQ/genética , Humanos , Interferón gamma/inmunología , Interleucina-15/genética , Masculino , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismoRESUMEN
Tripartite motif (TRIM) proteins mediate antiviral host defences by either directly targeting viral components or modulating innate immune responses. Here we identify a mechanism of antiviral restriction in which a TRIM E3 ligase controls viral replication by regulating the structure of host cell centrosomes and thereby nuclear lamina integrity. Through RNAi screening we identified several TRIM proteins, including TRIM43, that control the reactivation of Kaposi's sarcoma-associated herpesvirus. TRIM43 was distinguished by its ability to restrict a broad range of herpesviruses and its profound upregulation during herpesvirus infection as part of a germline-specific transcriptional program mediated by the transcription factor DUX4. TRIM43 ubiquitinates the centrosomal protein pericentrin, thereby targeting it for proteasomal degradation, which subsequently leads to alterations of the nuclear lamina that repress active viral chromatin states. Our study identifies a role of the TRIM43-pericentrin-lamin axis in intrinsic immunity, which may be targeted for therapeutic intervention against herpesviral infections.
Asunto(s)
Antígenos/metabolismo , Centrosoma/metabolismo , Infecciones por Herpesviridae/inmunología , Herpesvirus Humano 8/crecimiento & desarrollo , Proteínas de Motivos Tripartitos/metabolismo , Replicación Viral/fisiología , Células A549 , Animales , Línea Celular Tumoral , Chlorocebus aethiops , Células HEK293 , Células HeLa , Infecciones por Herpesviridae/prevención & control , Infecciones por Herpesviridae/virología , Proteínas de Homeodominio/metabolismo , Humanos , Lámina Nuclear/fisiología , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas de Motivos Tripartitos/genética , Proteínas de Motivos Tripartitos/fisiología , Ubiquitinación , Células Vero , Replicación Viral/genéticaRESUMEN
TRIM25 is a multi-domain, RING-type E3 ubiquitin ligase of the tripartite motif family that has important roles in multiple RNA-dependent processes. In particular, TRIM25 functions as an effector of RIG-I and ZAP, which are innate immune sensors that recognize viral RNA and induce ubiquitin-dependent anti-viral response mechanisms. TRIM25 is reported to also bind RNA, but the molecular details of this interaction or its relevance to anti-viral defense have not been elucidated. Here, we characterize the RNA-binding activity of TRIM25 and find that the protein binds both single-stranded and double-stranded RNA. Multiple regions of TRIM25 contribute to this functionality, including the C-terminal SPRY domain and a lysine-rich motif in the linker segment connecting the SPRY and coiled-coil domains. RNA binding modulates TRIM25's ubiquitination activity in vitro, its localization in cells, and its anti-viral activity. Taken together with other studies, our results indicate that RNA binding by TRIM25 has at least three important functional consequences: by enhancing ubiquitination activity, either through allosteric effects or through clustering of multiple TRIM25 molecules; by modulating the multi-domain structure of the TRIM25 dimer, and thereby structural coupling of the SPRY and RBCC elements during the ubiquitination reaction; and by facilitating subcellular localization of the E3 ligase during virus infection.
Asunto(s)
ARN Viral/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Proteínas de Motivos Tripartitos/química , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Virus/patogenicidad , Regulación Alostérica , Antivirales/química , Antivirales/metabolismo , Sitios de Unión , Virus del Dengue/genética , Virus del Dengue/patogenicidad , Células HEK293 , Células HeLa , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/patogenicidad , Unión Proteica , Dominios Proteicos , ARN Bicatenario/química , ARN Bicatenario/metabolismo , ARN Viral/química , Ubiquitinación , Virus/genéticaRESUMEN
Somatic mutations in tet methylcytosine dioxygenase 2 (TET2), which encodes an epigenetic modifier enzyme, drive the development of haematopoietic malignancies1-7. In both humans and mice, TET2 deficiency leads to increased self-renewal of haematopoietic stem cells with a net developmental bias towards the myeloid lineage1,4,8,9. However, pre-leukaemic myeloproliferation (PMP) occurs in only a fraction of Tet2-/- mice8,9 and humans with TET2 mutations1,3,5-7, suggesting that extrinsic non-cell-autonomous factors are required for disease onset. Here we show that bacterial translocation and increased interleukin-6 production, resulting from dysfunction of the small-intestinal barrier, are critical for the development of PMP in mice that lack Tet2 expression in haematopoietic cells. Furthermore, in symptom-free Tet2-/- mice, PMP can be induced by disrupting intestinal barrier integrity, or in response to systemic bacterial stimuli such as the toll-like receptor 2 agonist. PMP was reversed by antibiotic treatment and failed to develop in germ-free Tet2-/- mice, which illustrates the importance of microbial signals in the development of this condition. Our findings demonstrate the requirement for microbial-dependent inflammation in the development of PMP and provide a mechanistic basis for the variation in PMP penetrance observed in Tet2-/- mice. This study will prompt new lines of investigation that may profoundly affect the prevention and management of haematopoietic malignancies.
Asunto(s)
Enfermedades Asintomáticas , Fenómenos Fisiológicos Bacterianos , Proliferación Celular , Proteínas de Unión al ADN/deficiencia , Leucemia/microbiología , Leucemia/patología , Proteínas Proto-Oncogénicas/deficiencia , Animales , Infecciones Bacterianas/inmunología , Infecciones Bacterianas/microbiología , Fenómenos Fisiológicos Bacterianos/inmunología , Proteínas de Unión al ADN/genética , Dioxigenasas , Femenino , Vida Libre de Gérmenes , Inflamación/microbiología , Interleucina-6/inmunología , Mucosa Intestinal/metabolismo , Lactobacillus/química , Lactobacillus/citología , Lactobacillus/inmunología , Masculino , Ratones , Penetrancia , Permeabilidad , Proteínas Proto-Oncogénicas/genética , Receptor Toll-Like 2/agonistasRESUMEN
Autophagy and interferon (IFN)-mediated innate immunity are critical antiviral defence mechanisms, and recent evidence indicated that tripartite motif (TRIM) proteins are important regulators of both processes. Although the role of TRIM proteins in modulating antiviral cytokine responses has been well established, much less is known about their involvement in autophagy in response to different viral pathogens. Through a targeted RNAi screen examining the relevance of selected TRIM proteins in autophagy induced by herpes simplex virus 1 (HSV-1), encephalomyocarditis virus (EMCV) and influenza A virus (IAV), we identified several TRIM proteins that regulate autophagy in a virus-species-specific manner, as well as a few TRIM proteins that were essential for autophagy triggered by all three viruses and rapamycin, among them TRIM23. TRIM23 was critical for autophagy-mediated restriction of multiple viruses, and this activity was dependent on both its RING E3 ligase and ADP-ribosylation factor (ARF) GTPase activity. Mechanistic studies revealed that unconventional K27-linked auto-ubiquitination of the ARF domain is essential for the GTP hydrolysis activity of TRIM23 and activation of TANK-binding kinase 1 (TBK1) by facilitating its dimerization and ability to phosphorylate the selective autophagy receptor p62. Our work identifies the TRIM23-TBK1-p62 axis as a key component of selective autophagy and further reveals a role for K27-linked ubiquitination in GTPase-dependent TBK1 activation.
Asunto(s)
Autofagia , Proteínas de Unión al GTP/metabolismo , Interacciones Huésped-Patógeno , Proteínas Serina-Treonina Quinasas/metabolismo , Fenómenos Fisiológicos de los Virus , Virus de la Encefalomiocarditis/fisiología , Activación Enzimática , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/genética , Guanosina Trifosfato/metabolismo , Herpesvirus Humano 1/fisiología , Humanos , Hidrólisis , Inmunidad Innata , Virus de la Influenza A/fisiología , Fosforilación , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Transducción de Señal , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Viral infections have been proposed to elicit pathological processes leading to the initiation of T helper 1 (TH1) immunity against dietary gluten and celiac disease (CeD). To test this hypothesis and gain insights into mechanisms underlying virus-induced loss of tolerance to dietary antigens, we developed a viral infection model that makes use of two reovirus strains that infect the intestine but differ in their immunopathological outcomes. Reovirus is an avirulent pathogen that elicits protective immunity, but we discovered that it can nonetheless disrupt intestinal immune homeostasis at inductive and effector sites of oral tolerance by suppressing peripheral regulatory T cell (pTreg) conversion and promoting TH1 immunity to dietary antigen. Initiation of TH1 immunity to dietary antigen was dependent on interferon regulatory factor 1 and dissociated from suppression of pTreg conversion, which was mediated by type-1 interferon. Last, our study in humans supports a role for infection with reovirus, a seemingly innocuous virus, in triggering the development of CeD.
Asunto(s)
Antígenos/inmunología , Enfermedad Celíaca/inmunología , Enfermedad Celíaca/virología , Glútenes/inmunología , Inflamación/virología , Infecciones por Reoviridae/complicaciones , Infecciones por Reoviridae/inmunología , Células TH1/inmunología , Animales , Dieta/efectos adversos , Modelos Animales de Enfermedad , Ingeniería Genética , Humanos , Tolerancia Inmunológica , Inflamación/inmunología , Factor 1 Regulador del Interferón/genética , Factor 1 Regulador del Interferón/inmunología , Interferón Tipo I/genética , Interferón Tipo I/inmunología , Intestinos/inmunología , Intestinos/patología , Intestinos/virología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptor de Interferón alfa y beta/genética , Reoviridae/genéticaRESUMEN
An evaluation of CD4 T cell responses to candidate Chlamydia trachomatis vaccine antigens was conducted in an adolescent female cohort exposed through natural infection to explore antigen immunogenicity and correlation with protection from reinfection. The frequency of peripheral blood CD4 T cell IFN-γ and IL-17 responses to three candidate vaccine antigens, polymorphic membrane protein G (PmpG), F (PmpF), and major outer membrane protein (MOMP), were determined by ELISPOT; responses to chlamydial heat shock protein 60 (HSP60) and to elementary bodies (EB) were included for comparison. Responses of Infected (n=8), Seropositive/Uninfected (n=13), and Seronegative/Uninfected (n=18) participants were compared. The median CD4 IFN-γ response to EB was significantly increased in Infected (P=0.003) and Seropositive/Uninfected (P=0.002) versus Seronegative/Uninfected female subjects. Higher rates of positive IFN-γ responders to EB, PmpF, and MOMP were detected in Seropositive/Uninfected versus Seronegative/Uninfected participants (P=0.021). IL-17 responses were generally low. A positive IFN-γ response to any of the antigens tested was associated with a trend toward a reduced risk of reinfection, although not statistically significant. Among this adolescent cohort, chlamydial-specific CD4 IFN-γ but not IL-17 responses were detected in acutely and previously infected participants and a positive CD4 IFN-γ response was associated with a non-significant reduced risk of reinfection.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Linfocitos T CD4-Positivos/inmunología , Chlamydia trachomatis/inmunología , Interferón gamma/inmunología , Interleucina-17/inmunología , Adolescente , Adulto , Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Infecciones por Chlamydia/inmunología , Femenino , Humanos , Interferón gamma/biosíntesis , Interleucina-17/biosíntesis , Leucocitos Mononucleares/inmunología , Porinas/inmunología , Conducta Sexual , Adulto JovenRESUMEN
OBJECTIVE: Chlamydia trachomatis infections are a significant cause of reproductive tract pathology. Protective and pathological immune mediators must be differentiated to design a safe and effective vaccine. METHODS: Wild-type mice and mice deficient in IL-22 and IL-23 were infected intravaginally with Chlamydia muridarum, and their course of infection and oviduct pathology were compared. Local genital tract and draining lymph node immune responses were also examined in IL-23-deficient mice. RESULTS: IL-22- and IL-23-deficient mice exhibited normal susceptibility to infection and oviduct pathology. IL-23 was required for the development of a Chlamydia-specific Th17 response in the lymph nodes and for production of IL-22 and IL-17 in the genital tract. However, influx of Th1 and innate immune cells was not compromised in the absence of IL-23. CONCLUSION: IL-22 and IL-23 play either redundant or minimal roles in the pathogenesis of Chlamydia infection in the mouse model. Induction of Th17-associated cytokines by a Chlamydia vaccine should be avoided as these responses are not central to resolution of infection and have pathologic potential.
Asunto(s)
Infecciones por Chlamydia/inmunología , Chlamydia muridarum/inmunología , Interleucina-17/biosíntesis , Interleucina-23/inmunología , Interleucinas/biosíntesis , Infecciones del Sistema Genital/inmunología , Animales , Células Cultivadas , Infecciones por Chlamydia/microbiología , Infecciones por Chlamydia/patología , Femenino , Interleucina-17/inmunología , Interleucina-23/deficiencia , Interleucinas/deficiencia , Interleucinas/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oviductos/inmunología , Oviductos/patología , Infecciones del Sistema Genital/microbiología , Infecciones del Sistema Genital/patología , Interleucina-22RESUMEN
Resolution of Chlamydia genital tract infection is delayed in the absence of MyD88. In these studies, we first used bone marrow chimeras to demonstrate a requirement for MyD88 expression by hematopoietic cells in the presence of a wild-type epithelium. Using mixed bone marrow chimeras we then determined that MyD88 expression was specifically required in the adaptive immune compartment. Furthermore, adoptive transfer experiments revealed that CD4(+) T cell expression of MyD88 was necessary for normal resolution of genital tract infection. This requirement was associated with a reduced ability of MyD88(-/-)CD4(+) T cells to accumulate in the draining lymph nodes and genital tract when exposed to the same inflammatory milieu as wild-type CD4(+) T cells. We also demonstrated that the impaired infection control we observed in the absence of MyD88 could not be recapitulated by deficiencies in TLR or IL-1R signaling. In vitro, we detected an increased frequency of apoptotic MyD88(-/-)CD4(+) T cells upon activation in the absence of exogenous ligands for receptors upstream of MyD88. These data reveal an intrinsic requirement for MyD88 in CD4(+) T cells during Chlamydia infection and indicate that the importance of MyD88 extends beyond innate immune responses by directly influencing adaptive immunity.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Infecciones por Chlamydia/inmunología , Chlamydia muridarum/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Infecciones del Sistema Genital/inmunología , Traslado Adoptivo , Animales , Médula Ósea/inmunología , Linfocitos T CD4-Positivos/metabolismo , Infecciones por Chlamydia/microbiología , Femenino , Genitales Femeninos/citología , Genitales Femeninos/inmunología , Genitales Femeninos/microbiología , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/biosíntesis , Factor 88 de Diferenciación Mieloide/genética , Receptores de Interleucina-1/metabolismo , Infecciones del Sistema Genital/microbiologíaRESUMEN
The significant morbidities of ectopic pregnancy and infertility observed in women after Chlamydia trachomatis genital infection result from ascension of the bacteria from the endocervix to the oviduct, where an overly aggressive inflammatory response leads to chronic scarring and Fallopian tube obstruction. A vaccine to prevent chlamydia-induced disease is urgently needed. An important question for vaccine development is whether sterilizing immunity at the level of the oviduct is essential for protection because of the possibility that a chlamydial component drives a deleterious anamnestic T cell response upon oviduct reinfection. We show that mice inoculated with attenuated plasmid-cured strains of Chlamydia muridarum are protected from oviduct pathology upon challenge with wild-type C. muridarum Nigg despite induction of a response that did not prevent reinfection of the oviduct. Interestingly, repeated abbreviated infections with Nigg also elicited recall responses that protected the oviduct from pathology despite low-level reinfection of this vulnerable tissue site. Challenged mice displayed significant decreases in tissue infiltration of inflammatory leukocytes with marked reductions in frequencies of neutrophils but significant increases in frequencies of CD4 Th1 and CD8 T cells. An anamnestic antibody response was also detected. These data indicate that exposure to a live attenuated chlamydial vaccine or repeated abbreviated genital infection with virulent chlamydiae promotes anamnestic antibody and T cell responses that protect the oviduct from pathology despite a lack of sterilizing immunity at the site.
Asunto(s)
Infecciones por Chlamydia/inmunología , Chlamydia muridarum/inmunología , Enfermedades de los Genitales Femeninos/inmunología , Oviductos/patología , Animales , Vacunas Bacterianas , Infecciones por Chlamydia/microbiología , Infecciones por Chlamydia/patología , Femenino , Enfermedades de los Genitales Femeninos/microbiología , Enfermedades de los Genitales Femeninos/patología , Inflamación/metabolismo , Leucocitos/fisiología , Ratones , Ratones Endogámicos C3H , Oviductos/citología , Embarazo , Células TH1/fisiología , Factores de Tiempo , Vacunas AtenuadasRESUMEN
Our previous studies revealed that intravaginal infection of mice with a plasmid-deficient strain of Chlamydia muridarum, CM3.1, does not induce the development of oviduct pathology. In this study, we determined that infection with CM3.1 resulted in a significantly reduced frequency and absolute number of neutrophils in the oviducts during acute infection. This reduction in neutrophils was associated with significantly lower levels of neutrophil chemokines in the oviducts and decreased production of neutrophil chemokines by oviduct epithelial cells infected with CM3.1 in vitro. Infection with CM3.1 also resulted in an increased frequency of late apoptotic/dead neutrophils in the oviduct. Examination of the ability of Chlamydia trachomatis to prevent neutrophil apoptosis in vitro revealed that C. trachomatis strain D/UW-3/Cx exhibited an enhanced ability to prevent neutrophil apoptosis compared to plasmid-deficient CTD153, and this effect was dependent on the presence of CD14(high) monocytes. The presence of monocytes also resulted in enhanced neutrophil cytokine production and increased production of tissue-damaging molecules in response to D/UW-3/Cx relative to results with CTD153. Attempts to use antibody-mediated depletion to discern the specific role of neutrophils in infection control and pathology in vivo revealed that although Ly6G(high) neutrophils were eliminated from the blood and oviducts with this treatment, immature neutrophils and high levels of tissue-damaging molecules were still detectable in the upper genital tract. These data support the role of neutrophils in chlamydia-induced pathology and reveal that novel methods of depletion must be developed before their role can be specifically determined in vivo.