RESUMEN
Obesity-induced adipose tissue (AT) dysfunction results in a chronic low-grade inflammation that predisposes to the development of insulin resistance and type 2 diabetes. During the development of obesity, the AT-resident immune cell profile alters to create a pro-inflammatory state. Very recently, CD1d-restricted invariant (i) natural killer T (NKT) cells, a unique subset of lymphocytes that are reactive to so called lipid antigens, were implicated in AT homeostasis. Interestingly, recent data also suggest that human and mouse adipocytes can present such lipid antigens to iNKT cells in a CD1d-dependent fashion, but little is known about the lipid antigen presentation machinery in adipocytes. Here we show that CD1d, as well as the lipid antigen loading machinery genes pro-saposin (Psap), Niemann Pick type C2 (Npc2), α-galactosidase (Gla), are up-regulated in early adipogenesis, and are transcriptionally controlled by CCAAT/enhancer-binding protein (C/EBP)-ß and -δ. Moreover, adipocyte-induced Th1 and Th2 cytokine release by iNKT cells also occurred in the absence of exogenous ligands, suggesting the display of endogenous lipid antigen-D1d complexes by 3T3-L1 adipocytes. Furthermore, we identified microsomal triglyceride transfer protein, which we show is also under the transcriptional regulation of C/EBPß and -δ, as a novel player in the presentation of endogenous lipid antigens by adipocytes. Overall, our findings indicate that adipocytes can function as non-professional lipid antigen presenting cells, which may present an important aspect of adipocyte-immune cell communication in the regulation of whole body energy metabolism and immune homeostasis.
Asunto(s)
Adipocitos/inmunología , Adipocitos/metabolismo , Presentación de Antígeno , Antígenos CD1d/metabolismo , Proteínas Portadoras/metabolismo , Lípidos/inmunología , Células 3T3-L1 , Adipogénesis/genética , Adipogénesis/inmunología , Animales , Antígenos CD1d/genética , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteína delta de Unión al Potenciador CCAAT/genética , Proteína delta de Unión al Potenciador CCAAT/metabolismo , Proteínas Portadoras/genética , Comunicación Celular/inmunología , Metabolismo Energético , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Células T Asesinas Naturales/inmunología , Transcripción GenéticaRESUMEN
OBJECTIVE: Although genome wide association studies have partly uncovered the genetic basis of Crohn's disease (CD), it remains a challenge to link genetic polymorphisms to functional intestinal phenotypes. Paneth cells are specialised antimicrobial epithelial cells localised to the small-intestinal crypt-base. Here, we investigate whether genomic variations in ATG16L1 affect Paneth cell function. DESIGN: Genomic variation of ATG16L1 (T300A, rs2241880) was determined in DNA from 78 patients with CD and 12 healthy controls. Paraffin-embedded ileal biopsies from patients with genotype AA (n=17), GA (n=38) and patients with the GG allele (n=23) were stained for GRP78, phospho-EIF2α, lysozyme, cleaved-caspase 3, phosphohistone H3, phospho-IκB, p65, phospho-p38MAPK and PHLDA1. Microbial composition of biopsies was assessed by PCR. Disease phenotype was scored. RESULTS: In patients with quiescent disease but with an ATG16L1 risk allele, the endoplasmic reticulum (ER) stress markers GRP78 and pEIF2α were highly expressed in Paneth cells. Other CD risk gene variations did not correlate with Paneth cell ER stress. Functionally, patients with ER-stressed Paneth cells showed no changes in intestinal epithelial cells proliferation or apoptosis, Paneth cell or stem cell numbers, p65, phospho-IκB and phospho-p38 staining. However, a significantly increased presence of adherent-invasive Escherichia coli was observed in biopsies from patients with ER-stressed Paneth cells. Phenotypically, patients with GRP78 positive Paneth cells have relatively less colonic disease over ileal disease (-21%, p=0.04), more fistulas (+21%, p=0.05) and an increased need for intestinal surgery (+38%, p=0.002). CONCLUSIONS: The ATG16L1 T300A polymorphism defines a specific subtype of patients with CD, characterised by Paneth cell ER stress even during quiescent disease. Paneth cell ER stress correlates with bacterial persistence, and is thus likely to modulate antimicrobial functionality of this cell type in patients with CD.
Asunto(s)
Proteínas Portadoras/genética , Enfermedad de Crohn/genética , Estrés del Retículo Endoplásmico/genética , Predisposición Genética a la Enfermedad , Células de Paneth/metabolismo , Polimorfismo de Nucleótido Simple , Proteínas Relacionadas con la Autofagia , Biopsia , Estudios de Casos y Controles , Células Cultivadas , Enfermedad de Crohn/metabolismo , Enfermedad de Crohn/microbiología , Enfermedad de Crohn/patología , ADN Bacteriano/análisis , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/fisiología , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Marcadores Genéticos , Técnicas de Genotipaje , Humanos , Íleon/metabolismo , Íleon/microbiología , Íleon/patología , Microbiota , Análisis Multivariante , Células de Paneth/microbiología , Células de Paneth/patología , Fenotipo , Factores de RiesgoRESUMEN
Cross-presentation of IgG-containing immune complexes (ICs) is an important means by which dendritic cells (DCs) activate CD8(+) T cells, yet it proceeds by an incompletely understood mechanism. We show that monocyte-derived CD8(-)CD11b(+) DCs require the neonatal Fc receptor for IgG (FcRn) to conduct cross-presentation of IgG ICs. Consequently, in the absence of FcRn, Fcγ receptor (FcγR)-mediated antigen uptake fails to initiate cross-presentation. FcRn is shown to regulate the intracellular sorting of IgG ICs to the proper destination for such cross-presentation to occur. We demonstrate that FcRn traps antigen and protects it from degradation within an acidic loading compartment in association with the rapid recruitment of key components of the phagosome-to-cytosol cross-presentation machinery. This unique mechanism thus enables cross-presentation to evolve from an atypically acidic loading compartment. FcRn-driven cross-presentation is further shown to control cross-priming of CD8(+) T-cell responses in vivo such that during chronic inflammation, FcRn deficiency results in inadequate induction of CD8(+) T cells. These studies thus demonstrate that cross-presentation in CD8(-)CD11b(+) DCs requires a two-step mechanism that involves FcγR-mediated internalization and FcRn-directed intracellular sorting of IgG ICs. Given the centrality of FcRn in controlling cross-presentation, these studies lay the foundation for a unique means to therapeutically manipulate CD8(+) T-cell responses.
Asunto(s)
Reactividad Cruzada/inmunología , Células Dendríticas/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Inmunoglobulina G/inmunología , Receptores Fc/inmunología , Animales , Antígenos/inmunología , Western Blotting , Antígeno CD11b/inmunología , Antígeno CD11b/metabolismo , Antígenos CD8/inmunología , Antígenos CD8/metabolismo , Colitis/inducido químicamente , Colitis/inmunología , Colitis/metabolismo , Citosol/inmunología , Citosol/metabolismo , Células Dendríticas/metabolismo , Sulfato de Dextran , Citometría de Flujo , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Concentración de Iones de Hidrógeno , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mutación , NADPH Oxidasa 2 , NADPH Oxidasas/inmunología , NADPH Oxidasas/metabolismo , Fagosomas/inmunología , Fagosomas/metabolismo , Unión Proteica , Receptores Fc/genética , Receptores Fc/metabolismo , Receptores de IgG/inmunología , Receptores de IgG/metabolismo , ATPasas de Translocación de Protón Vacuolares/inmunología , ATPasas de Translocación de Protón Vacuolares/metabolismo , Proteínas de Unión al GTP rab/inmunología , Proteínas de Unión al GTP rab/metabolismo , Proteínas rab27 de Unión a GTPRESUMEN
Dendritic cells (DCs) and monocyte-derived macrophages (MΦs) are key components of intestinal immunity. However, the lack of surface markers differentiating MΦs from DCs has hampered understanding of their respective functions. Here, we demonstrate that, using CD64 expression, MΦs can be distinguished from DCs in the intestine of both mice and humans. On that basis, we revisit the phenotype of intestinal DCs in the absence of contaminating MΦs and we delineate a developmental pathway in the healthy intestine that leads from newly extravasated Ly-6C(hi) monocytes to intestinal MΦs. We determine how inflammation impacts this pathway and show that T cell-mediated colitis is associated with massive recruitment of monocytes to the intestine and the mesenteric lymph node (MLN). There, these monocytes differentiate into inflammatory MΦs endowed with phagocytic activity and the ability to produce inducible nitric oxide synthase. In the MLNs, inflammatory MΦs are located in the T-cell zone and trigger the induction of proinflammatory T cells. Finally, T cell-mediated colitis develops irrespective of intestinal DC migration, an unexpected finding supporting an important role for MLN-resident inflammatory MΦs in the etiology of T cell-mediated colitis.
Asunto(s)
Colitis/inmunología , Células Dendríticas/inmunología , Mucosa Intestinal/inmunología , Ganglios Linfáticos/inmunología , Macrófagos/inmunología , Mesenterio/inmunología , Receptores de IgG/inmunología , Células TH1/inmunología , Animales , Antígenos Ly/inmunología , Diferenciación Celular/inmunología , Colitis/patología , Células Dendríticas/patología , Humanos , Inmunidad Mucosa , Mucosa Intestinal/patología , Ganglios Linfáticos/patología , Macrófagos/patología , Mesenterio/patología , Ratones , Ratones Noqueados , Monocitos/inmunología , Monocitos/patología , Células TH1/patologíaRESUMEN
Xenotoxic damage in inflammatory diseases, including IBD (inflammatory bowel disease), is compounded by reduced activity of the xenobiotic transporter ABCG2 (ATP-binding-cassette G2) during the inflammatory state. An association between the activation of the unfolded protein response pathway and inflammation prompted us to investigate the possibility that reduced ABCG2 activity is causally linked to this response. To this end, we correlated expression of ABCG2 and the unfolded protein response marker GRP78 (glucose-regulated protein of 78 kDa) in colon biopsies from healthy individuals (n=9) and patients with inactive (n=67) or active (n=55) IBD, ischaemic colitis (n=10) or infectious colitis (n=14). In addition, tissue specimens throughout the small bowel from healthy individuals (n=27) and from patients with inactive (n=9) or active (n=25) Crohn's disease were co-stained for ABCG2 and GRP78. In all biopsies from patients with active inflammation, irrespective of the underlying disease, an absolute negative correlation was observed between epithelial ABCG2 expression and GRP78 expression, suggesting that inflammation-dependent activation of the unfolded protein response is responsible for suppression of ABCG2 function. The link between the unfolded protein response and functional ABCG2 expression was further corroborated by live imaging of ABCG2-expressing cells, which showed that various inflammatory mediators, including nitric oxide, activate the unfolded protein response and concomitantly reduce plasma membrane localization as well as transport function of ABCG2. Thus a novel mechanism for explaining xenobiotic stress during inflammation emerges in which intestinal inflammation activates the unfolded protein response, in turn abrogating defences against xenobiotic challenge by impairing ABCG2 expression and function.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Colitis/metabolismo , Enfermedad de Crohn/metabolismo , Regulación de la Expresión Génica/fisiología , Mucosa Intestinal/metabolismo , Proteínas de Neoplasias/metabolismo , Pliegue de Proteína , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Colitis/clasificación , Chaperón BiP del Retículo Endoplásmico , Células Epiteliales/metabolismo , Células HEK293 , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Inflamación/metabolismo , Mucosa Intestinal/citología , Mucosa Intestinal/patología , Proteínas de Neoplasias/genéticaRESUMEN
Ulcerative colitis (UC) is associated with a high risk of developing colorectal cancer (CRC). The mechanisms by which chronic inflammatory responses in the colon may promote CRC remain only partially understood, but may involve reduced negative regulation of interleukin (IL)-6 signaling towards signal transducer and activator of transcription 3 activation through the loss of SOCS3 expression, unleashing the full carcinogenic potential of this transcription factor. Thus, we analyzed SOCS3 expression in the colon of healthy controls, as well as in a cohort of UC patients with varying degrees of dysplasia. We observe that the loss of epithelial SOCS3 expression delimits the areas subject to dysplasia in UC, suggesting an important tumour-suppressive role of SOCS3 downregulation, early in the transformation process. Importantly, methylation of the SOCS3 promotor appears to constitute an important regulatory mechanism for colonic SOCS3 expression as SOCS3 methylation status in CRC cells correlates with a disability to upregulate SOCS3 upon IL-6 stimulation, whereas forced demethylation using 5-aza-2'-deoxycytidine restores SOCS3 expression and inhibits IL-6-induced p-signal transducer and activator of transcription 3 activation and proliferation. Expression of the DNA methyltransferase gene DMNT1 is prominent in dysplastic cells and correlates with low or absent SOCS3 expression. Thus, induction of DNMT1 expression in the chronically inflamed colon may release IL-6 signaling towards signal transducer and activator of transcription 3 from inhibition through SOCS3 increasing the propensity to malignant transformation. Hence, DNMT1 emerges as a rational target in preventive strategies aimed at counteracting UC-CRC.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Colitis Ulcerosa/metabolismo , Neoplasias Colorrectales/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Interleucina-6/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Colitis Ulcerosa/patología , Colon/metabolismo , Neoplasias Colorrectales/patología , ADN (Citosina-5-)-Metiltransferasa 1 , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Humanos , Mucosa Intestinal/metabolismo , Masculino , Proteína 3 Supresora de la Señalización de CitocinasRESUMEN
Ulcerative colitis (UC) is a chronic disease associated with long periods of quiescent disease followed by fulminant exacerbation. Imminent relapse in UC is associated with high mucosal expression of suppressor of cytokine signaling 3 (SOCS3); hence, knowledge of the mechanisms driving mucosal SOCS3 expression may provide important clues as to rational therapy. Thus, here we aim to characterize the molecular forces driving SOCS3 expression in the mucosal compartment, focusing on druggable pathways. The colon epithelial cell line Caco-2 was stimulated with interferon (IFN)-γ, interleukin (IL)-4 or prostaglandin E(2) (PGE(2)) to allow correlations between SOCS3 expression with signal transducer and activator of transcription 1 (STAT1), STAT6 and adenosine 3',5'-cyclic monophosphate (cAMP) signaling, respectively. The physiological relevance of the findings obtained was assessed by immunohistochemical staining for the activated forms of STAT1, STAT6, protein kinase A (PKA)-Cγ and cAMP response element-binding protein (CREB) in biopsies from inactive UC patients and controls. Stimulation with IFN-γ, IL-4 or PGE(2) induced activation of STAT1, STAT6 and cAMP, respectively, in colonic cells, without any signs of concomitant STAT3 activation. Forced activation of all these signaling pathways was sufficient for SOCS3 expression. Biopsies from patients with inactive UC showed significant increase of phosphorylated STAT1 (p-STAT1) (p < 0.0001), p-STAT6 (p = 0.0001), p-PKA-Cγ (p = 0.0003) and p-CREB (p = 0.0025) expression compared with controls. STAT3-independent SOCS3 induction in inactive UC involves multiple proinflammatory signaling pathways and contradicts the usefulness of pathway-specific antiinflammatory drugs for preventing relapse. Our findings suggest that broad-spectrum antiinflammatory drugs are essential to counteract increases in SOCS3 expression and exacerbation of disease. Our results highlight the multifactorial nature of the factors that cause exacerbation in UC.
Asunto(s)
Colitis Ulcerosa/metabolismo , AMP Cíclico/metabolismo , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT6/metabolismo , Transducción de Señal , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Adulto , Células CACO-2 , Colitis Ulcerosa/enzimología , Colitis Ulcerosa/patología , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismo , Citocinas/farmacología , Enterocitos/efectos de los fármacos , Enterocitos/enzimología , Enterocitos/patología , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Transducción de Señal/efectos de los fármacos , Proteína 3 Supresora de la Señalización de CitocinasRESUMEN
BACKGROUND: Perinatal arterial ischaemic stroke (PAIS) is an important cause of neurodevelopmental disabilities. In this first-in-human study, we aimed to assess the feasibility and safety of intranasally delivered bone marrow-derived allogeneic mesenchymal stromal cells (MSCs) to treat PAIS in neonates. METHODS: In this open-label intervention study in collaboration with all neonatal intensive care units in the Netherlands, we included neonates born at full term (≥36 weeks of gestation) with MRI-confirmed PAIS in the middle cerebral artery region. All eligible patients were transferred to the neonatal intensive care unit of the Wilhelmina Children's Hospital. Neonates received one dose of 45-50â×â106 bone-marrow derived MSCs intranasally within 7 days of presenting signs of PAIS. The primary endpoints were acute and subacute safety outcomes, including vital signs, blood markers, and the occurrence of toxicity, adverse events, and serious adverse events. The occurrence of unexpected cerebral abnormalities by a repeat MRI at 3 months of age was a secondary endpoint. As part of standard clinical follow-up at Wilhelmina Children's Hospital, we assessed corticospinal tract development on MRI and performed motor assessments at 4 months of age. This study is registered with ClinicalTrials.gov, NCT03356821. FINDINGS: Between Feb 11, 2020, and April 29, 2021, ten neonates were enrolled in the study. Intranasal administration of MSCs was well tolerated in all ten neonates. No serious adverse events were observed. One adverse event was seen: a mild transient fever of 38°C without the need for clinical intervention. Blood inflammation markers (C-reactive protein, procalcitonin, and leukocyte count) were not significantly different pre-administration versus post-administration and, although thrombocyte levels increased (p=0·011), all were within the physiological range. Follow-up MRI scans did not show unexpected structural cerebral abnormalities. All ten patients had initial pre-Wallerian changes in the corticospinal tracts, but only four (40%) patients showed asymmetrical corticospinal tracts at follow-up MRI. Abnormal early motor assessment was found in three (30%) infants. INTERPRETATION: This first-in-human study demonstrates that intranasal bone marrow-derived MSC administration in neonates after PAIS is feasible and no serious adverse events were observed in patients followed up until 3 months of age. Future large-scale placebo-controlled studies are needed to determine the therapeutic effect of intranasal MSCs for PAIS. FUNDING: Netherlands Organization for Health Research and Development (ZonMw).
Asunto(s)
Isquemia Encefálica , Accidente Cerebrovascular Isquémico , Células Madre Mesenquimatosas , Accidente Cerebrovascular , Niño , Estudios de Factibilidad , Humanos , Lactante , Recién Nacido , Países Bajos , Investigación , Accidente Cerebrovascular/diagnóstico por imagen , Accidente Cerebrovascular/terapia , Resultado del TratamientoRESUMEN
The intestinal tract is covered by a total of 300 square metres of IECs (intestinal epithelial cells) that covers the entire intestinal mucosa. For protection against luminal xenobiotics, pathogens and commensal microbes, these IECs are equipped with membrane-bound transporters as well as the ability to secrete specific protective proteins. In patients with active IBD (inflammatory bowel disease), the expression of these proteins, e.g. ABC (ATP-binding cassette) transporters such as ABCG2 (ABC transporter G2) and defensins, is decreased, thereby limiting the protection against various luminal threats. Correct ER (endoplasmic reticulum)-dependent protein folding is essential for the localization and function of secreted and membrane-bound proteins. Inflammatory triggers, such as cytokines and nitric oxide, can impede protein folding, which causes the accumulation of unfolded proteins inside the ER. As a result, the unfolded protein response is activated which can lead to a cellular process named ER stress. The protein folding impairment affects the function and localization of several proteins, including those involved in protection against xenobiotics. In the present review, we discuss the possible inflammatory pathways affecting protein folding and eventually leading to IEC malfunction in patients with active IBD.
Asunto(s)
Enfermedades Inflamatorias del Intestino/metabolismo , Pliegue de Proteína , Animales , Colitis/tratamiento farmacológico , Colitis/metabolismo , Colitis/fisiopatología , Retículo Endoplásmico/fisiología , Células Epiteliales/metabolismo , Humanos , Mucosa Intestinal/patología , Estrés Fisiológico , Respuesta de Proteína DesplegadaRESUMEN
BACKGROUND: Mouse models have shown that interleukin (IL)6 stimulates survival, proliferation and progression to cancer of intestinal epithelial cells via activation of signal transducers and activators of transcription 3 (STAT3). OBJECTIVE: To investigate the expression of IL6/phosphorylated STAT3 (p-STAT3)/suppressor of cytokine signalling 3 (SOCS3) in biopsy specimens from patients with ulcerative colitis (UC) and UC-related colorectal cancer (CRC) progression. METHODS: Biopsy specimens from patients with inactive UC (n=18), active UC (n=28), UC with low-grade dysplasia (LGD) (n=9), UC with high-grade dysplasia (HGD) (n=7), UC-CRC (n=11) and sporadic CRC (n=14) were included. Biopsy specimens (n=9) from patients without colonic abnormalities served as control. The protein expression of IL6, p-STAT3 and SOCS3 was determined immunohistochemically. RESULTS: Patients with active UC had significantly more IL6 and p-STAT3-positive epithelial cells than both patients with inactive UC and controls (strong positive IL6: 53.6%, 11.1% and 0%, respectively; p-STAT3: 64.3%, 22.2% and 11.1%, respectively; all pAsunto(s)
Transformación Celular Neoplásica/metabolismo
, Colitis Ulcerosa/metabolismo
, Neoplasias Colorrectales/metabolismo
, Interleucina-6/metabolismo
, Adulto
, Colitis Ulcerosa/complicaciones
, Colon/metabolismo
, Neoplasias Colorrectales/etiología
, Metilación de ADN
, ADN de Neoplasias/genética
, Progresión de la Enfermedad
, Células Epiteliales/metabolismo
, Femenino
, Humanos
, Técnicas para Inmunoenzimas
, Mucosa Intestinal/metabolismo
, Masculino
, Persona de Mediana Edad
, Factor de Transcripción STAT3/metabolismo
, Transducción de Señal/fisiología
, Proteína 3 Supresora de la Señalización de Citocinas
, Proteínas Supresoras de la Señalización de Citocinas/genética
, Proteínas Supresoras de la Señalización de Citocinas/metabolismo
, Adulto Joven
RESUMEN
Hematopoietic cell transplantation (HCT) is a last resort, potentially curative treatment option for pediatric patients with refractory acute myeloid leukemia (AML). Cord blood transplantation (CBT) results in less relapses and less graft-versus-host disease when compared to other sources. Nevertheless, still more than half of the children die from relapses. We therefore designed a strategy to prevent relapses by inducing anti-AML immunity after CBT, using a CB-derived dendritic cell (CBDC) vaccine generated from CD34+ CB cells from the same graft. We here describe the optimization and validation of good manufacturing practice (GMP)-grade production of the CBDC vaccine. We show the feasibility of expanding low amounts of CD34+ cells in a closed bag system to sufficient DCs per patient for at least three rounds of vaccinations. The CBDCs showed upregulated costimulatory molecules after maturation and showed enhanced CCR7-dependent migration toward CCL19 in a trans-well migrations assay. CBDCs expressed Wilms' tumor 1 (WT1) protein after electroporation with WT1-mRNA, but were not as potent as CBDCs loaded with synthetic long peptides (peptivator). The WT1-peptivator loaded CBDCs were able to stimulate T-cells both in a mixed lymphocyte reaction as well as in an antigen-specific (autologous) setting. The autologous stimulated T-cells lysed not only the WT1+ cell line, but most importantly, also primary pediatric AML cells. Altogether, we provide a GMP-protocol of a highly mature CBDC vaccine, loaded with WT1 peptivator and able to stimulate autologous T-cells in an antigen-specific manner. Finally, these T-cells lysed primary pediatric AML demonstrating the competence of the CBDC vaccine strategy.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Sangre Fetal/citología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/inmunología , Proteínas WT1/genética , Presentación de Antígeno , Biomarcadores , Vacunas contra el Cáncer/uso terapéutico , Terapia Combinada , Trasplante de Células Madre de Sangre del Cordón Umbilical , Citotoxicidad Inmunológica , Humanos , Inmunofenotipificación , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/terapia , Activación de Linfocitos/inmunología , Prueba de Cultivo Mixto de Linfocitos , Pronóstico , Recurrencia , Especificidad del Receptor de Antígeno de Linfocitos T , Linfocitos T/inmunología , Linfocitos T/metabolismo , Resultado del Tratamiento , Proteínas WT1/inmunologíaRESUMEN
BACKGROUND: The adjuvant activity of air pollution particles on allergic airway sensitization is well known, but the cellular mechanisms underlying this adjuvant potential are not clear. OBJECTIVE: We sough to study the role of dendritic cells and the costimulatory molecules CD80 and CD86 in the adjuvant activity of ultrafine carbon black particles (CBP). METHODS: The proliferation of CFSE-labeled DO11.10 CD4 cells was studied after intranasal exposure to particles and ovalbumin (OVA). Next the frequency of myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells and their expression of CD80 and CD86 were studied in the peribronchial lymph nodes (PBLNs). The expression of costimulatory molecules was also studied on bone marrow-derived mDCs after exposure to CBPs in vitro, and the importance of costimulation in CBP adjuvant activity was assessed by using CD80/CD86-deficient mice or cytotoxic T lymphocyte-associated antigen 4 (CTLA4)-Ig in vivo. RESULTS: Our data show that CBPs plus OVA caused proliferation of DO11.10 CD4 cells and high levels of cytokine production in the PBLNs. Furthermore, the combined CBP plus OVA exposure increased the number of mDCs and expression of costimulatory molecules in the PBLNs. In addition, CBPs upregulated the expression of CD80/CD86 molecules on dendritic cells in vitro, which are necessary for the particle adjuvant effects in vivo. CONCLUSION: Together this study shows the importance of dendritic cells and costimulation in particle adjuvant activity. Furthermore, we show for the first time that CBPs can also directly induce maturation of dendritic cells.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Células Dendríticas/efectos de los fármacos , Material Particulado/inmunología , Hipersensibilidad Respiratoria/inducido químicamente , Hollín/farmacología , Traslado Adoptivo , Animales , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Linfocitos T CD4-Positivos/inmunología , Proliferación Celular , Citocinas/biosíntesis , Células Dendríticas/citología , Células Dendríticas/inmunología , Femenino , Citometría de Flujo , Fluoresceínas , Pulmón/citología , Pulmón/inmunología , Ganglios Linfáticos/inmunología , Ratones , Ovalbúmina/inmunología , Tamaño de la Partícula , Hipersensibilidad Respiratoria/inmunología , SuccinimidasRESUMEN
BACKGROUND: The Pregnane X Receptor (PXR) is a principal signal transducer in mucosal responses to xenobiotic stress. It is well-recognized that inflammatory bowel disease is accompanied by xenobiotic stress, but the importance of the PXR in limiting inflammatory responses in inflammatory bowel disease remains obscure at best. METHODS: We stimulate a total of 106 colonic biopsies from 19 Crohn's disease patients with active disease, 36 colonic biopsies from 8 control patients, colonic organoids and various cell culture models (either proficient or genetically deficient with respect to PXR) in vitro with the PXR ligand rifampicin or vehicle. Effects on NF-κB activity are assessed by measuring interleukin-8 (IL-8) and interleukin-1ß (IL-1ß) mRNA levels by qPCR and in cell culture models by NF-κB reporter-driven luciferase activity and Western blot for signal transduction elements. RESULTS: We observe a strict inverse correlation between colonic epithelial PXR levels and NF-κB target gene expression in colonic biopsies from Crohn's disease patients. PXR, activated by rifampicin, is rate-limiting for mucosal NF-κB activation in IBD. The correlation between colonic epithelial PXR levels and NF-κB target gene expression was also observed in intestinal organoids system. Furthermore, in preclinical in vitro models of intestinal inflammation, including intestinal organoids, genetic inactivation of PXR unleashes NF-κB-dependent signal transduction whereas conversely NF-κB signaling reduces levels of PXR expression. CONCLUSIONS: Our data indicate that the PXR is a major and clinically relevant antagonist of NF-κB activity in the intestinal epithelial compartment during inflammatory bowel disease.
Asunto(s)
Enfermedades Inflamatorias del Intestino/metabolismo , Mucosa Intestinal/metabolismo , FN-kappa B/metabolismo , Receptor X de Pregnano/metabolismo , Biopsia , Línea Celular Tumoral , Citocinas/metabolismo , Humanos , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Rifampin/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Dendritic cells (DCs) are professional antigen-presenting cells which instruct both the innate and adaptive immune systems. Once mature, they have the capacity to activate and prime naïve T cells for recognition and eradication of pathogens and tumor cells. These characteristics make them excellent candidates for vaccination strategies. Most DC vaccines have been generated from ex vivo culture of monocytes (mo). The use of mo-DCs as vaccines to induce adaptive immunity against cancer has resulted in clinical responses but, overall, treatment success is limited. The application of primary DCs or DCs generated from CD34⺠stem cells have been suggested to improve clinical efficacy. Cord blood (CB) is a particularly rich source of CD34⺠stem cells for the generation of DCs, but the dynamics and plasticity of the specific DC lineage development are poorly understood. Using flow sorting of DC progenitors from CB cultures and subsequent RNA sequencing, we found that CB-derived DCs (CB-DCs) exclusively originate from CD115âº-expressing progenitors. Gene set enrichment analysis displayed an enriched conventional DC profile within the CD115-derived DCs compared with CB mo-DCs. Functional assays demonstrated that these DCs matured and migrated upon good manufacturing practice (GMP)-grade stimulation and possessed a high capacity to activate tumor-antigen-specific T cells. In this study, we developed a culture protocol to generate conventional DCs from CB-derived stem cells in sufficient numbers for vaccination strategies. The discovery of a committed DC precursor in CB-derived stem cell cultures further enables utilization of conventional DC-based vaccines to provide powerful antitumor activity and long-term memory immunity.
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BACKGROUND: Natural killer T (NKT) cells in adipose tissue (AT) contribute to whole body energy homeostasis. RESULTS: Inhibition of the glucosylceramide synthesis in adipocytes impairs iNKT cell activity. CONCLUSION: Glucosylceramide biosynthesis pathway is important for endogenous lipid antigen activation of iNKT cells in adipocytes. SIGNIFICANCE: Unraveling adipocyte-iNKT cell communication may help to fight obesity-induced AT dysfunction. Overproduction and/or accumulation of ceramide and ceramide metabolites, including glucosylceramides, can lead to insulin resistance. However, glucosylceramides also fulfill important physiological functions. They are presented by antigen presenting cells (APC) as endogenous lipid antigens via CD1d to activate a unique lymphocyte subspecies, the CD1d-restricted invariant (i) natural killer T (NKT) cells. Recently, adipocytes have emerged as lipid APC that can activate adipose tissue-resident iNKT cells and thereby contribute to whole body energy homeostasis. Here we investigate the role of the glucosylceramide biosynthesis pathway in the activation of iNKT cells by adipocytes. UDP-glucose ceramide glucosyltransferase (Ugcg), the first rate limiting step in the glucosylceramide biosynthesis pathway, was inhibited via chemical compounds and shRNA knockdown in vivo and in vitro. ß-1,4-Galactosyltransferase (B4Galt) 5 and 6, enzymes that convert glucosylceramides into potentially inactive lactosylceramides, were subjected to shRNA knock down. Subsequently, (pre)adipocyte cell lines were tested in co-culture experiments with iNKT cells (IFNγ and IL4 secretion). Inhibition of Ugcg activity shows that it regulates presentation of a considerable fraction of lipid self-antigens in adipocytes. Furthermore, reduced expression levels of either B4Galt5 or -6, indicate that B4Galt5 is dominant in the production of cellular lactosylceramides, but that inhibition of either enzyme results in increased iNKT cell activation. Additionally, in vivo inhibition of Ugcg by the aminosugar AMP-DNM results in decreased iNKT cell effector function in adipose tissue. Inhibition of endogenous glucosylceramide production results in decreased iNKT cells activity and cytokine production, underscoring the role of this biosynthetic pathway in lipid self-antigen presentation by adipocytes.
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Adipocitos/metabolismo , Glucosilceramidas/biosíntesis , Células T Asesinas Naturales/metabolismo , Adipocitos/citología , Presentación de Antígeno , Comunicación Celular , Línea Celular , Técnicas de Cocultivo , Citocinas/metabolismo , Glucosilceramidas/metabolismo , Humanos , Resistencia a la Insulina , Lípidos/inmunología , Activación de Linfocitos , Células T Asesinas Naturales/citologíaRESUMEN
BACKGROUND: Although it is well recognized that fatigue is an important problem in many of the quiescent inflammatory bowel disease (IBD) patients, it is unknown whether the immune status is different in fatigued versus non-fatigued patients. In this study, we contrasted various characteristics of the immune system in fatigued against non-fatigued patients with IBD in clinical remission. PATIENTS AND METHODS: Patients with IBD in clinical remission were phenotyped according to the Montreal classification, and the checklist individual strength-fatigue (CIS-fatigue) was used to assess fatigue (CIS-fatigue ≥ 35). Flow cytometry on peripheral blood samples was used to investigate differences in leukocyte subsets. The expression of various cytokines was determined in stimulated whole blood and serum samples using enzyme-linked immunosorbent assay. Differences between fatigued and non-fatigued patients with IBD were assessed. RESULTS: In total, 55 patients were included in the fatigue group (FG) and 29 patients in the non-fatigue group (NFG). No differences in demographic and clinical characteristics were observed between the groups. Flow cytometry data showed a significantly lower percentage of monocytes (p = 0.011) and a higher percentage of memory T-cells (p = 0.005) and neutrophils (p = 0.033) in the FG compared with the NFG. Whole blood stimulation showed increased TNF-α (p = 0.022) and IFN-γ (p = 0.047) in the FG. The median serum level was significantly higher for IL-12 (p < 0.001) and IL-10 (p = 0.005) and lower for IL-6 (p = 0.002) in the FG compared with NFG. CONCLUSION: Significant differences in immune profile between fatigued and non-fatigued patients with IBD in clinical remission were found, which point out to a chronically active and Th1-skewed immune system in patients with fatigue. Whether these immune differences are directly involved in the fatigue complaints via immune-to-brain communication pathways remains to be determined. As such, further exploration of the underlying immune effects associated with fatigue is warranted to determine potential treatment options.
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BACKGROUND: SH2 domain containing inositol-5-phosphatase (SHIP1) is an important modulator of innate and adaptive immunity. In mice, loss of SHIP1 provokes severe ileitis resembling Crohn's disease (CD), as a result of deregulated immune responses, altered cytokine production and intestinal fibrosis. Recently, SHIP1 activity was shown to be correlated to the presence of a CD-associated single nucleotide polymorphism in ATG16L1. Here, we studied SHIP1 activity and expression in an adult cohort of CD patients. METHODS: SHIP1 activity, protein and mRNA expression in peripheral blood mononuclear cells from CD patients in clinical remission were determined by Malachite green assay, Western blotting and qRT-PCR respectively. Genomic DNA was genotyped for ATG16L1 rs2241880. RESULTS: SHIP1 protein levels are profoundly diminished in a subset of patients; however, SHIP1 activity and expression are not correlated to ATG16L1 SNP status in this adult cohort. CONCLUSIONS: Aberrant SHIP1 activity can contribute to disease in a subset of adult CD patients, and warrants further investigation.
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Proteínas Relacionadas con la Autofagia/genética , Enfermedad de Crohn/genética , Regulación hacia Abajo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/metabolismo , Polimorfismo de Nucleótido Simple , Adulto , Línea Celular , Estudios de Cohortes , Enfermedad de Crohn/metabolismo , Femenino , Regulación de la Expresión Génica , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
To gain more insight into the mechanisms of particulate matter (PM)-induced adjuvant activity, we studied the kinetics of airway toxicity/inflammation and allergic sensitization to ovalbumin (OVA) in response to ultrafine carbon black particles (CBP). Mice were exposed intranasally to OVA alone or in combination with different concentrations of CBP. Airway toxicity and inflammation were assessed at days 4 and 8. Immune adjuvant effects were studied in the lung draining peribronchial lymph nodes (PBLN) at day 8. Antigen-specific IgE was measured at days 21 and 28, whereas allergic airway inflammation was studied after OVA challenges (day 28). Results show that a total dose of 200 microg CBP per mouse, but not 20 microg or 2 microg, induced immediate airway inflammation. This 200 microg CBP was the only dose that had immune adjuvant activity, by inducing enlargement of the PBLN and increasing OVA-specific production of Th2 cytokines (IL-4, IL-5, and IL-10). The immune adjuvant activity of 200 microg CBP dosing was further examined. Whereas increased OVA-specific IgE levels in serum on day 21 confirms systemic sensitization, this was further supported by allergic airway inflammation after challenges with OVA. Our data show a link between early airway toxicity and adjuvant effects of CBP. In addition, results indicate that local cytokine production early after exposure to CBP is predictive of allergic airway inflammation. In addition this model appears suitable for studying the role of airway toxicity, inflammation and other mechanisms of particle adjuvant activity, and predicting the adjuvant potential of different particles.
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Carbono/toxicidad , Inflamación/inducido químicamente , Hipersensibilidad Respiratoria/inducido químicamente , Enfermedades Respiratorias/inducido químicamente , Administración Intranasal , Animales , Asma/inmunología , Asma/patología , Líquido del Lavado Bronquioalveolar/citología , Carbono/administración & dosificación , Relación Dosis-Respuesta a Droga , Femenino , Citometría de Flujo , Inmunoglobulina E/inmunología , Inflamación/patología , Pulmón/inmunología , Pulmón/patología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Recuento de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/inmunología , Hipersensibilidad Respiratoria/patología , Enfermedades Respiratorias/patología , Células Th2/efectos de los fármacos , Células Th2/inmunologíaRESUMEN
The poor survival rates of refractory/relapsed acute myeloid leukemia (AML) patients after haematopoietic cell transplantation (HCT) requires the development of additional immune therapeutic strategies. As the elicitation of tumor-antigen specific cytotoxic T lymphocytes (CTLs) is associated with reduced relapses and enhanced survival, enhanced priming of these CTLs using an anti-AML vaccine may result in long-term immunity against AML. Cord blood (CB), as allogeneic HCT source, may provide a unique setting for such post-HCT vaccination, considering its enhanced graft-versus-leukemia (GvL) effects and population of highly responsive naïve T cells. It is our goal to develop a powerful and safe immune therapeutic strategy composed of CB-HCT followed by vaccination with CB CD34+-derived dendritic cells (DCs) presenting the oncoprotein Wilms Tumor-1 (WT1), which is expressed in AML-blasts in the majority of patients. Here, we describe the optimization of a clinically applicable DC culture protocol. This two-step protocol consisting of an expansion phase followed by the differentiation toward DCs, enables us to generate sufficient cord blood-derived DCs (CBDCs) in the clinical setting. At the end of the culture, the CBDCs exhibit a mature surface phenotype, are able to migrate, express tumor antigen (WT1) after electroporation with mRNA encoding the full-length WT1 protein, and stimulate WT1-specific T cells.