RESUMEN
Poly(ADP-ribose) polymerases (PARPs) synthesize and bind branched polymers of ADP-ribose to acceptor proteins using NAD as a substrate and participate in the control of gene transcription and DNA repair. PARP1, the most abundant isoform, regulates the expression of proinflammatory mediator cytokines, chemokines, and adhesion molecules, and inhibition of PARP1 enzymatic activity reduced or ameliorated autoimmune diseases in several experimental models, including colitis. However, the mechanism(s) underlying the protective effects of PARP1 inhibition in colitis and the cell types in which Parp1 deletion has the most significant impact are unknown. The objective of the current study was to determine the impact of Parp1 deletion on the innate immune response to mucosal injury and on the gut microbiome composition. Parp1 deficiency was evaluated in DSS-induced colitis in WT, Parp1(-/-), Rag2(-/-), and Rag2(-/-)×Parp1(-/-) double knock-out mice. Genome-wide analysis of the colonic transcriptome and fecal 16S amplicon profiling was performed. Compared with WT, we demonstrated that Parp1(-/-) were protected from dextran-sulfate sodium-induced colitis and that this protection was associated with a dramatic transcriptional reprogramming in the colon. PARP1 deficiency was also associated with a modulation of the colonic microbiota (increases relative abundance of Clostridia clusters IV and XIVa) and a concomitant increase in the frequency of mucosal CD4(+)CD25(+) Foxp3(+) regulatory T cells. The protective effects conferred by Parp1 deletion were lost in Rag2(-/-) × Parp1(-/-) mice, highlighting the role of the adaptive immune system for full protection.
Asunto(s)
Inmunidad Adaptativa , Colitis/inmunología , Colon/inmunología , Inmunidad Innata , Inmunidad Mucosa , Mucosa Intestinal/inmunología , Poli(ADP-Ribosa) Polimerasas/deficiencia , Animales , Colitis/inducido químicamente , Colitis/genética , Colitis/patología , Colon/lesiones , Colon/patología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Sulfato de Dextran/toxicidad , Mucosa Intestinal/lesiones , Mucosa Intestinal/patología , Ratones , Ratones Noqueados , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patologíaRESUMEN
Primarily defined by their antigen-presenting property, dendritic cells (DCs) are being implemented as cancer vaccines in immunotherapeutic interventions. DCs can also function as direct tumor cell killers. How DC cytotoxic activity can be efficiently harnessed and the mechanisms controlling this nonconventional property are not fully understood. We report here that the tumoricidal potential of mouse DCs generated from myeloid precursors with GM-CSF and IL-15 (IL-15 DCs) can be triggered with the Toll-like receptor (TLR) 4 ligand lipopolysaccharide to a similar extent compared with that of their counterparts, conventionally generated with IL-4 (IL-4 DCs). The mechanism of tumor cell killing depends on the induction of iNOS expression by DCs. In contrast, interferon (IFN)-γ induces the cytotoxic activity of IL-4 but not IL-15 DCs. Although the IFN-γ-STAT-1 signaling pathway is overall functional in IL-15 DCs, IFN-γ fails to induce iNOS expression in these cells. iNOS expression is negatively controlled in IFN-γ-stimulated IL-15 DCs by the cooperation between the E3 SUMO ligase PIAS1 and STAT-3, and can be partially restored with PIAS1 siRNA and STAT-3 inhibitors.
Asunto(s)
Células Dendríticas/metabolismo , Interferón gamma/metabolismo , Interleucina-15/metabolismo , Proteínas Inhibidoras de STAT Activados/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Línea Celular Tumoral , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Interleucina-4/metabolismo , Ligandos , Lipopolisacáridos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Factor de Transcripción STAT1/metabolismo , Transducción de Señal/fisiología , Receptor Toll-Like 4/metabolismoRESUMEN
OBJECTIVE: Children with congenital heart disease have loss of intestinal epithelial barrier function, which increases their risk for postoperative sepsis and organ dysfunction. We do not understand how postoperative cardiopulmonary support or the inflammatory response to cardiopulmonary bypass might alter intestinal epithelial barrier function. We examined variation in a panel of plasma biomarkers to reflect intestinal epithelial barrier function (cellular and paracellular) after cardiopulmonary bypass and in response to routine ICU care. DESIGN: Prospective cohort. SETTING: University medical center cardiac ICU. PATIENTS: Twenty children aged between newborn and 18 years undergoing repair or palliation of congenital heart disease with cardiopulmonary bypass. INTERVENTIONS: We measured baseline and repeated plasma intestinal fatty acid-binding protein, citrulline, claudin 3, and dual sugar permeability testing to reflect intestinal epithelial integrity, epithelial function, paracellular integrity, and paracellular function, respectively. We measured baseline and repeated plasma proinflammatory (interleukin-6, tumor necrosis factor-α, and interferon-γ) and anti-inflammatory (interleukin-4 and interleukin-10) cytokines, known to modulate intestinal epithelial barrier function in murine models of cardiopulmonary bypass. MEASUREMENTS AND MAIN RESULTS: All patients had abnormal baseline intestinal fatty acid-binding protein concentrations (mean, 3,815.5 pg/mL; normal, 41-336 pg/mL). Cytokine response to cardiopulmonary bypass was associated with early, but not late, changes in plasma concentrations of intestinal fatty acid-binding protein 2 and citrulline. Variation in biomarker concentrations over time was associated with aspects of ICU care indicating greater severity of illness: claudin 3, intestinal fatty acid-binding protein 2, and dual sugar permeability test ratio were associated with symptoms of feeding intolerance (p < 0.05), whereas intestinal fatty acid-binding protein was positively associated with vasoactive-inotrope score (p = 0.04). Citrulline was associated with larger arteriovenous oxygen saturation difference (p = 0.04) and had a complex relationship with vasoactive-inotrope score. CONCLUSIONS: Children undergoing cardiopulmonary bypass for repair or palliation of congenital heart disease are at risk for intestinal injury and often present with evidence for loss of intestinal epithelial integrity preoperatively. Greater severity of illness requiring increased cardiopulmonary support rather than the inflammatory response to cardiopulmonary bypass seems to mediate late postoperative intestinal epithelial barrier function.
Asunto(s)
Biomarcadores/sangre , Citocinas/sangre , Cardiopatías Congénitas/cirugía , Enfermedades Intestinales/etiología , Mucosa Intestinal/patología , Niño , Preescolar , Estudios de Cohortes , Femenino , Humanos , Lactante , Inflamación , Unidades de Cuidado Intensivo Pediátrico , Enfermedades Intestinales/sangre , Masculino , Periodo Posoperatorio , Estudios ProspectivosRESUMEN
BACKGROUND & AIMS: Dysregulated Ca(2+) homeostasis likely contributes to the etiology of inflammatory bowel disease-associated loss of bone mineral density. Experimental colitis leads to decreased expression of Klotho, a protein that supports renal Ca(2+) reabsorption by stabilizing the transient receptor potential vanilloid 5 (TRPV5) channel on the apical membrane of distal tubule epithelial cells. METHODS: Colitis was induced in mice via administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS) or transfer of CD4(+)interleukin-10(-/-) and CD4(+), CD45RB(hi) T cells. We investigated changes in bone metabolism, renal processing of Ca(2+), and expression of TRPV5. RESULTS: Mice with colitis had normal serum levels of Ca(2+) and parathormone. Computed tomography analysis showed a decreased density of cortical and trabecular bone, and there was biochemical evidence for reduced bone formation and increased bone resorption. Increased fractional urinary excretion of Ca(2+) was accompanied by reduced levels of TRPV5 protein in distal convoluted tubules, with a concomitant increase in TRPV5 sialylation. In mouse renal intermedullary collecting duct epithelial (mIMCD3) cells transduced with TRPV5 adenovirus, the inflammatory cytokines tumor necrosis factor, interferon-γ, and interleukin-1ß reduced levels of TRPV5 on the cell surface, leading to its degradation. Cytomix induced interaction between TRPV5 and UBR4 (Ubiquitin recoginition 4), an E3 ubiquitin ligase; knockdown of UBR4 with small interfering RNAs prevented cytomix-induced degradation of TRPV5. The effects of cytokines on TRPV5 were not observed in cells stably transfected with membrane-bound Klotho; TRPV5 expression was preserved when colitis was induced with TNBS in transgenic mice that overexpressed Klotho or in mice with T-cell transfer colitis injected with soluble recombinant Klotho. CONCLUSIONS: After induction of colitis in mice via TNBS administration or T-cell transfer, tumor necrosis factor and interferon-γ reduced the expression and activity of Klotho, which otherwise would protect TRPV5 from hypersialylation and cytokine-induced TRPV5 endocytosis, UBR4-dependent ubiquitination, degradation, and urinary wasting of Ca(2+).
Asunto(s)
Densidad Ósea , Canales de Calcio/metabolismo , Calcio/metabolismo , Colitis/metabolismo , Riñón/metabolismo , Procesamiento Proteico-Postraduccional , Canales Catiónicos TRPV/metabolismo , Animales , Biomarcadores/metabolismo , Linfocitos T CD4-Positivos/trasplante , Colitis/inducido químicamente , Colitis/inmunología , Glucuronidasa/metabolismo , Interferón gamma/metabolismo , Proteínas Klotho , Ratones , Ratones Transgénicos , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tomografía Computarizada por Rayos X , Ácido Trinitrobencenosulfónico , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
In vitro data and transgenic mouse models suggest a role for TGF-ß signaling in dendritic cells (DCs) to prevent autoimmunity primarily through maintenance of DCs in their immature and tolerogenic state characterized by low expression of MHC class II (MHCII) and costimulatory molecules and increased expression of IDO, among others. To test whether a complete lack of TGF-ß signaling in DCs predisposes mice to spontaneous autoimmunity and to verify the mechanisms implicated previously in vitro, we generated conditional knockout (KO) mice with Cre-mediated DC-specific deletion of Tgfbr2 (DC-Tgfbr2 KO). DC-Tgfbr2 KO mice die before 15 wk of age with multiorgan autoimmune inflammation and spontaneous activation of T and B cells. Interestingly, there were no significant differences in the expression of MHCII, costimulatory molecules, or IDO in secondary lymphoid organ DCs, although Tgfbr2-deficient DCs were more proinflammatory in vitro and in vivo. DC-Tgfbr2 KO showed attenuated Foxp3 expression in regulatory T cells (Tregs) and abnormal expansion of CD25(-)Foxp3(+) Tregs in vivo. Tgfbr2-deficient DCs secreted elevated levels of IFN-γ and were not capable of directing Ag-specific Treg conversion unless in the presence of anti-IFN-γ blocking Ab. Adoptive transfer of induced Tregs into DC-Tgfbr2 KO mice partially rescued the phenotype. Therefore, in vivo, TGF-ß signaling in DCs is critical in the control of autoimmunity through both Treg-dependent and -independent mechanisms, but it does not affect MHCII and costimulatory molecule expression.
Asunto(s)
Enfermedades Autoinmunes/prevención & control , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Proteínas Serina-Treonina Quinasas/deficiencia , Receptores de Factores de Crecimiento Transformadores beta/deficiencia , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Animales , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/patología , Colitis/genética , Colitis/inmunología , Colitis/prevención & control , Células Dendríticas/patología , Modelos Animales de Enfermedad , Tolerancia Inmunológica/genética , Inmunofenotipificación , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , Proteínas Serina-Treonina Quinasas/fisiología , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Linfocitos T Reguladores/patologíaRESUMEN
Chronic inflammation and enteric infections are frequently associated with epithelial Na(+)/H(+) exchange (NHE) inhibition. Alterations in electrolyte transport and in mucosal pH associated with inflammation may represent a key mechanism leading to changes in the intestinal microbial composition. NHE3 expression is essential for the maintenance of the epithelial barrier function. NHE3(-/-) mice develop spontaneous distal chronic colitis and are highly susceptible to dextran sulfate (DSS)-induced mucosal injury. Spontaneous colitis is reduced with broad-spectrum antibiotics treatment, thus highlighting the importance of the microbiota composition in NHE3 deficiency-mediated colitis. We herein characterized the colonic microbiome of wild-type (WT) and NHE3(-/-) mice housed in a conventional environment using 454 pyrosequencing. We demonstrated a significant decrease in the phylogenetic diversity of the luminal and mucosal microbiota of conventional NHE3(-/-) mice compared with WT. Rederivation of NHE3(-/-) mice from conventional to a barrier facility eliminated the signs of colitis and decreased DSS susceptibility. Reintroduction of the conventional microflora into WT and NHE3(-/-) mice from the barrier facility resulted in the restoration of the symptoms initially described in the conventional environment. Interestingly, qPCR analysis of the microbiota composition in mice kept in the barrier facility compared with reconventionalized mice showed a significant reduction of Clostridia classes IV and XIVa. Therefore, the gut microbiome plays a prominent role in the pathogenesis of colitis in NHE3(-/-) mice, and, reciprocally, NHE3 also plays a critical role in shaping the gut microbiota. NHE3 deficiency may be a critical contributor to dysbiosis observed in patients with inflammatory bowel disease.
Asunto(s)
Bacterias/clasificación , Colitis/microbiología , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Colitis/inducido químicamente , Colitis/genética , Sulfato de Dextran/toxicidad , Heces/microbiología , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/genéticaRESUMEN
Dendritic cells (DCs) encompass a heterogeneous population of cells capable of orchestrating innate and adaptive immune responses. The ability of DCs to act as professional APCs has been the foundation for the development and use of these cells as vaccines in cancer immunotherapy. DCs are also endowed with the nonconventional property of directly killing tumor cells. The current study investigates the regulation of murine DC cytotoxic function by T lymphocytes. We provide evidence that CD4(+) Th-1, but not Th-2, Th-17 cells, or regulatory T cells, are capable of inducing DC cytotoxic function. IFN-γ was identified as the major factor responsible for Th-1-induced DC tumoricidal activity. Tumor cell killing mediated by Th-1-activated killer DCs was dependent on inducible NO synthase expression and NO production. Importantly, Th-1-activated killer DCs were capable of presenting the acquired Ags from the killed tumor cells to T lymphocytes in vitro or in vivo. These observations offer new possibilities for the application of killer DCs in cancer immunotherapy.
Asunto(s)
Citotoxicidad Inmunológica , Células Dendríticas/inmunología , Interferón gamma/fisiología , Neoplasias Mamarias Experimentales/inmunología , Melanoma Experimental/inmunología , Receptores de Interferón/fisiología , Células TH1/inmunología , Células TH1/metabolismo , Animales , Presentación de Antígeno/genética , Presentación de Antígeno/inmunología , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Línea Celular Tumoral , Técnicas de Cocultivo , Citotoxicidad Inmunológica/genética , Células Dendríticas/metabolismo , Femenino , Interferón gamma/metabolismo , Neoplasias Mamarias Experimentales/patología , Neoplasias Mamarias Experimentales/terapia , Melanoma Experimental/patología , Melanoma Experimental/terapia , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores de Interferón/deficiencia , Receptores de Interferón/genética , Receptor de Interferón gammaRESUMEN
Curcumin (diferulolylmethane) is an anti-inflammatory phenolic compound found effective in preclinical models of inflammatory bowel diseases (IBD) and in ulcerative colitis patients. Pharmacokinetics of curcumin and its poor systemic bioavailability suggest that it targets preferentially intestinal epithelial cells. The intestinal epithelium, an essential component of the gut innate defense mechanisms, is profoundly affected by IFN-γ, which can disrupt the epithelial barrier function, prevent epithelial cell migration and wound healing, and prime epithelial cells to express major histocompatibility complex class II (MHC-II) molecules and to serve as nonprofessional antigen-presenting cells. In this report we demonstrate that curcumin inhibits IFN-γ signaling in human and mouse colonocytes. Curcumin inhibited IFN-γ-induced gene transcription, including CII-TA, MHC-II genes (HLA-DRα, HLA-DPα1, HLA-DRß1), and T cell chemokines (CXCL9, 10, and 11). Acutely, curcumin inhibited Stat1 binding to the GAS cis-element, prevented Stat1 nuclear translocation, and reduced Jak1 phosphorylation and phosphorylation of Stat1 at Tyr(701). Longer exposure to curcumin led to endocytic internalization of IFNγRα followed by lysosomal fusion and degradation. In summary, curcumin acts as an IFN-γ signaling inhibitor in colonocytes with biphasic mechanisms of action, a phenomenon that may partially account for the beneficial effects of curcumin in experimental colitis and in human IBD.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Colon/efectos de los fármacos , Curcumina/farmacología , Interferón gamma/antagonistas & inhibidores , Mucosa Intestinal/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Células Cultivadas , Quimiocinas/efectos de los fármacos , Humanos , Mucosa Intestinal/inmunología , Janus Quinasa 1/metabolismo , Complejo Mayor de Histocompatibilidad , Ratones , Fosforilación , Factor de Transcripción STAT1/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
BACKGROUND & AIMS: Klotho (KL) is an anti-inflammatory protein that protects the endothelium from nitric oxide (NO)-induced dysfunction, reduces the expression of endothelial adhesion molecules, and potentially regulates T-cell functions. KL deficiency leads to premature senescence and impaired Ca2+/Pi homeostasis, which can lead to inflammatory bowel disease (IBD)-associated osteopenia/osteoporosis. We investigated the changes in renal expression of Kl as a consequence of colitis. METHODS: We studied 3 mouse models of IBD: colitis induced by trinitrobenzene sulfonic acid, colitis induced by microflora (in gnotobiotic interleukin-10(-/-)), and colitis induced by adoptive transfer of CD4(+)CD45RB(high) T cells. Effects of the tumor necrosis factor (TNF) and interferon (IFN)-gamma on Kl expression and the activity of its promoter were examined in renal epithelial cells (mpkDCT4 and mIMCD3). RESULTS: Renal expression of Kl messenger RNA (mRNA) and protein was reduced in all 3 models of IBD. Reduced level of KL correlated with the severity of colitis; the effect was reversed by neutralizing antibodies against TNF. In vitro, TNF inhibited Kl expression, an effect potentiated by IFN-gamma. The combination of TNF and IFN-gamma increased expression of inducible nitric oxide synthase (iNOS) and increased NO production. The effect of IFN-gamma was reproduced by exposure to an NO donor and reversed by the iNOS inhibitor. In cells incubated with TNF and/or IFN-gamma, Kl mRNA stability was unaffected, whereas Kl promoter activity was reduced, indicating that these cytokines regulate Kl at the transcriptional level. CONCLUSIONS: The down-regulation of KL that occurs during inflammation might account for the extraintestinal complications such as abnormalities in bone homeostasis that occur in patients with IBD.
Asunto(s)
Colitis/metabolismo , Glucuronidasa/antagonistas & inhibidores , Interferón gamma/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Traslado Adoptivo , Animales , Calcio/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Glucuronidasa/genética , Enfermedades Inflamatorias del Intestino/complicaciones , Enfermedades Inflamatorias del Intestino/metabolismo , Interleucina-10/fisiología , Proteínas Quinasas JNK Activadas por Mitógenos/fisiología , Riñón/metabolismo , Proteínas Klotho , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Óxido Nítrico/fisiología , Osteoporosis/etiología , Transcripción Genética/efectos de los fármacosRESUMEN
Chimeric antigen receptor (CAR) T cells mediate potent antigen-specific antitumor activity; however, their indirect effects on the endogenous immune system are not well characterized. Remarkably, we demonstrate that CAR T-cell treatment of mouse syngeneic glioblastoma (GBM) activates intratumoral myeloid cells and induces endogenous T-cell memory responses coupled with feed-forward propagation of CAR T-cell responses. IFNγ production by CAR T cells and IFNγ responsiveness of host immune cells are critical for tumor immune landscape remodeling to promote a more activated and less suppressive tumor microenvironment. The clinical relevance of these observations is supported by studies showing that human IL13Rα2-CAR T cells activate patient-derived endogenous T cells and monocytes/macrophages through IFNγ signaling and induce the generation of tumor-specific T-cell responses in a responding patient with GBM. These studies establish that CAR T-cell therapy has the potential to shape the tumor microenvironment, creating a context permissible for eliciting endogenous antitumor immunity. SIGNIFICANCE: Our findings highlight the critical role of IFNγ signaling for a productive CAR T-cell therapy in GBM. We establish that CAR T cells can activate resident myeloid populations and promote endogenous T-cell immunity, emphasizing the importance of host innate and adaptive immunity for CAR T-cell therapy of solid tumors.This article is highlighted in the In This Issue feature, p. 2113.
Asunto(s)
Glioblastoma/tratamiento farmacológico , Inmunoterapia Adoptiva , Interferón gamma/metabolismo , Células Mieloides/inmunología , Receptores Quiméricos de Antígenos/inmunología , Animales , Glioblastoma/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Microambiente Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND & AIMS: NHE3 is a target of inhibition by proinflammatory cytokines and pathogenic bacteria, an event contributing to diarrhea in infectious and idiopathic colitis. In mice, NHE3 deficiency leads to mild diarrhea, increased intestinal expression of interferon (IFN)-gamma, and distal colitis, suggesting its role in epithelial barrier homeostasis. Our aim was to investigate the role of NHE3 in maintaining mucosal integrity. METHODS: Control or dextran sulfate sodium (DSS)-treated, 6- to 8-week-old wild-type (WT) and NHE3(-/-) mice were used for the experiments. Small intestines were dissected for further analysis. RESULTS: NHE3(-/-) mice have elevated numbers of CD8alpha(+) T and natural killer cells in the intraepithelial lymphocytes and lamina propria lymphocytes compartments, representing the source of IFN-gamma. NHE3(-/-) mice display alterations in epithelial gene and protein expression patterns that predispose them to a high susceptibility to DSS, with accelerated mortality resulting from intestinal bleeding, hypovolemic shock, and sepsis, even at a very low DSS concentration. Microarray analysis and intestinal hemorrhage indicate that NHE3 deficiency predisposes mice to DSS-induced small intestinal injury, a segment never reported as affected by DSS, and demonstrate major differences in the colonic response to DSS challenge in WT and NHE3(-/-) mice. In NHE3(-/-) mice, broad-spectrum oral antibiotics or anti-asialo GM1 antibodies reduce the expression of IFN-gamma and iNOS to basal levels and delay but do not prevent severe mortality in response to DSS treatment. CONCLUSIONS: These results suggest that NHE3 participates in mucosal responses to epithelial damage, acting as a modifier gene determining the extent of the gut inflammatory responses in the face of intestinal injury.
Asunto(s)
Sulfato de Dextran/toxicidad , Homeostasis , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Intercambiadores de Sodio-Hidrógeno/fisiología , Animales , Recuento de Células Sanguíneas , Colon/metabolismo , Regulación hacia Abajo , Endotelina-1/metabolismo , Gangliósido G(M1)/metabolismo , Hemorragia Gastrointestinal/inducido químicamente , Interferón gamma/metabolismo , Mucosa Intestinal/efectos de los fármacos , Intestino Delgado/efectos de los fármacos , Intestino Delgado/metabolismo , Intestino Delgado/patología , Ratones , Ratones Noqueados , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Transducción de Señal , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/genéticaRESUMEN
Sarcomas are heterogeneous malignant mesenchymal neoplasms with limited sensitivity to immunotherapy. We recently demonstrated an increase in Kynurenine Pathway (KP) activity in the plasma of sarcoma patients treated with pembrolizumab. While the KP has already been described to favor immune escape through the degradation of L-Tryptophan and production of metabolites including L-Kynurenine, Indoleamine 2,3 dioxygenase (IDO1), a first rate-limiting enzyme of the KP, still represents an attractive therapeutic target, and its blockade had not yet been investigated in sarcomas. Using immunohistochemistry, IDO1 and CD8, expression profiles were addressed within 203 cases of human sarcomas. At a preclinical level, we investigated the modulation of the KP upon PDL1 blockade in a syngeneic model of sarcoma through mRNA quantification of key KP enzymes within the tumor. Furthermore, in order to evaluate the possible anti-tumor effect of IDO blockade in combination with PDL1 blockade, an innovative IDO inhibitor (GDC-0919) was used. Its effect was first assessed on Kynurenine to Tryptophan ratio at plasmatic level and also within the tumor. Following GDC-0919 treatment, alone or in combination with anti-PDL1 antibody, tumor growth, immune cell infiltration, and gene expression profiling were measured. IDO1 expression was observed in 39.1% of human sarcoma cases and was significantly higher in tumors with high CD8 infiltration. In the pre-clinical setting, blockade of PDL1 led to a strong anti-tumor effect and was associated with an intratumoral inflammatory cytokines signature driven by Ifng but also with a modulation of the KP enzymes including Ido1 and Ido2. IDO1 inhibition using GDC-0919 resulted in (i) a significant decrease of plasmatic Kynurenine to Tryptophan ratio and in (ii) a decrease of tumoral Kynurenine. However, GDC-0919 used alone or combined with anti-PDL1, did not show anti-tumoral activity and did not affect the tumor immune cell infiltrate. In order to elucidate the mechanism(s) underlying the lack of effect of GDC-0919, we analyzed the gene expression profile of intratumoral biopsies. Interestingly, we have found that GDC-0919 induced a downregulation of the expression of pvr and granzymes, and an upregulation of inhba and Dtx4 suggesting a potential role of the IDO pathway in the control of NK function.
Asunto(s)
Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Sarcoma/tratamiento farmacológico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Femenino , Humanos , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Indolamina-Pirrol 2,3,-Dioxigenasa/análisis , Indolamina-Pirrol 2,3,-Dioxigenasa/fisiología , Quinurenina/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Persona de Mediana Edad , Sarcoma/inmunología , Sarcoma/metabolismo , Células Tumorales Cultivadas , Adulto JovenRESUMEN
Inflammatory bowel diseases (IBD) are associated with functional inhibition of epithelial Na+/H+ exchange. In mice, a selective disruption of NHE3 (Slc9a3), a major apical Na+/H+ exchanger, also promotes IBD-like symptoms and gut microbial dysbiosis. We hypothesized that disruption of Na+/H+ exchange is necessary for the development of dysbiosis, which promotes an exacerbated mucosal inflammatory response. Therefore, we performed a temporal analysis of gut microbiota composition, and mucosal immune response to adoptive T cell transfer was evaluated in Rag2-/- and NHE3-/-/Rag2-/- (DKO) mice with and without broad-spectrum antibiotics. Microbiome (16S profiling), colonic histology, T cell and neutrophil infiltration, mucosal inflammatory tone, and epithelial permeability were analyzed. In adoptive T cell transfer colitis model, Slc9a3 status was the most significant determinant of gut microbial community. In DKO mice, NHE3-deficiency and dysbiosis were associated with dramatically accelerated and exacerbated disease, with rapid body weight loss, increased mucosal T cell and neutrophil influx, increased mucosal cytokine expression, increased permeability, and expansion of CD25-FoxP3+ Tregs; this enhanced susceptibility was alleviated by oral broad-spectrum antibiotics. Based on these results and our previous work, we postulate that epithelial electrolyte homeostasis is an important modulator in the progression of colitis, acting through remodeling of the gut microbial community.
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Colitis/inmunología , Intestinos/microbiología , Intercambiadores de Sodio-Hidrógeno/metabolismo , Linfocitos T/inmunología , Animales , Colitis/metabolismo , Ratones , Ratones NoqueadosRESUMEN
BACKGROUND: Intestinal microbiota influences the progression of colitis-associated colorectal cancer. With diet being a key determinant of the gut microbial ecology, dietary interventions are an attractive avenue for the prevention of colitis-associated colorectal cancer. Curcumin is the most active constituent of the ground rhizome of the Curcuma longa plant, which has been demonstrated to have anti-inflammatory, antioxidative, and antiproliferative properties. METHODS: Il10 mice on 129/SvEv background were used as a model of colitis-associated colorectal cancer. Starting at 10 weeks of age, wild-type or Il10 mice received 6 weekly intraperitoneal injections of azoxymethane (AOM) or phosphate-buffered saline (PBS) and were started on either a control or a curcumin-supplemented diet. Stools were collected every 4 weeks for microbial community analysis. Mice were killed at 30 weeks of age. RESULTS: Curcumin-supplemented diet increased survival, decreased colon weight/length ratio, and, at 0.5%, entirely eliminated tumor burden. Although colonic histology indicated improvement with curcumin, no effects of mucosal immune responses have been observed in PBS/Il10 mice and limited effects were seen in AOM/Il10 mice. In wild-type and in Il10 mice, curcumin increased bacterial richness, prevented age-related decrease in alpha diversity, increased the relative abundance of Lactobacillales, and decreased Coriobacterales order. Taxonomic profile of AOM/Il10 mice receiving curcumin was more similar to those of wild-type mice than those fed control diet. CONCLUSIONS: In AOM/Il10 model, curcumin reduced or eliminated colonic tumor burden with limited effects on mucosal immune responses. The beneficial effect of curcumin on tumorigenesis was associated with the maintenance of a more diverse colonic microbial ecology.
Asunto(s)
Transformación Celular Neoplásica/efectos de los fármacos , Colon/patología , Neoplasias Colorrectales/tratamiento farmacológico , Curcumina/administración & dosificación , Mucosa Intestinal/patología , Microbiota/efectos de los fármacos , Animales , Azoximetano/administración & dosificación , Carcinógenos/farmacología , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Colon/microbiología , Neoplasias Colorrectales/inducido químicamente , Suplementos Dietéticos , Modelos Animales de Enfermedad , Inmunidad Mucosa , Ratones , Ratones de la Cepa 129 , Ratones NoqueadosRESUMEN
Despite the wide range of available therapies, human colon cancers remain difficult to cure. Evidence for efficient antitumoral immune responses to be raised is now widely accepted, and numerous strategies exploiting the host immune system have been developed. A treatment based on the lipid-A derivative OM-174 has been developed in our laboratory. OM-174 induces the rejection of tumors established by injection of PROb colon cancer cells in syngeneic BDIX rats. Our immunohistochemistry study demonstrated that OM-174 treatment is associated with tumor cell apoptosis. Caspase 3 activation was detected 24 h after the first OM-174 injection. Six days after the beginning of the treatment, dendritic cells were the first immune cells that invaded tumor nodules. When dendritic cells came into contact with apoptotic tumor cells, an increased expression of the costimulatory molecules B7-1 and B7-2 was detected at the surface of these cells. Five days later, macrophages were found in the tumor nodules. Lymphocytes organized into a crown surrounding the nodules that progressively regressed during the treatment. T lymphocytes were not in contact with tumor cells or apoptotic cells at any time point. The kinetics of tumor cell apoptosis induced by OM-174, as well as the appearance of innate followed by adaptative immune cells in the tumor nodules, were compatible with cell activation and the development of immune response.
Asunto(s)
Neoplasias del Colon/tratamiento farmacológico , Lípido A/uso terapéutico , Lipopolisacáridos/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Caspasa 3 , Caspasas/metabolismo , División Celular/efectos de los fármacos , Neoplasias del Colon/inmunología , Neoplasias del Colon/patología , Cinética , Activación de Linfocitos/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Masculino , Trasplante de Neoplasias , Ratas , Ratas Endogámicas , Trasplante IsogénicoRESUMEN
Myeloid-derived suppressor cells (MDSC) expand in tumor-bearing hosts and play a central role in cancer immune evasion by inhibiting adaptive and innate immunity. They therefore represent a major obstacle for successful cancer immunotherapy. Different strategies have thus been explored to deplete and/or inactivate MDSC in vivo. Using a murine mammary cancer model, we demonstrated that doxorubicin selectively eliminates MDSC in the spleen, blood, and tumor beds. Furthermore, residual MDSC from doxorubicin-treated mice exhibited impaired suppressive function. Importantly, the frequency of CD4(+) and CD8(+) T lymphocytes and consequently the effector lymphocytes or natural killer (NK) to suppressive MDSC ratios were significantly increased following doxorubicin treatment of tumor-bearing mice. In addition, the proportion of NK and cytotoxic T cell (CTL) expressing perforin and granzyme B and of CTL producing IFN-γ was augmented by doxorubicin administration. Of therapeutic relevance, this drug efficiently combined with Th1 or Th17 lymphocytes to suppress tumor development and metastatic disease. MDSC isolated from patients with different types of cancer were also sensitive to doxorubicin-mediated cytotoxicity in vitro. These results thus indicate that doxorubicin may be used not only as a direct cytotoxic drug against tumor cells, but also as a potent immunomodulatory agent that selectively impairs MDSC-induced immunosuppression, thereby fostering the efficacy of T-cell-based immunotherapy.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Doxorrubicina/farmacología , Inmunoterapia Adoptiva/métodos , Células Mieloides/efectos de los fármacos , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Animales , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Células Mieloides/inmunología , Células Mieloides/patologíaRESUMEN
MDSCs and Tregs play an essential role in the immunosuppressive networks that contribute to tumor-immune evasion. The mechanisms by which tumors promote the expansion and/or function of these suppressive cells and the cross-talk between MDSC and Treg remain incompletely defined. Previous reports have suggested that MDSC may contribute to Treg induction in cancer. Herein, we provide evidence that tumor-induced gr-MDSCs, endowed with the potential of suppressing conventional T Lc, surprisingly impair TGF-ß1-mediated generation of CD4(+)CD25(+)FoxP3(+) iTregs. Furthermore, gr-MDSCs impede the proliferation of nTregs without, however, affecting FoxP3 expression. Suppression of iTreg differentiation from naïve CD4(+) cells by gr-MDSC occurs early in the polarization process, requires inhibition of early T cell activation, and depends on ROS and IDO but does not require arginase 1, iNOS, NO, cystine/cysteine depletion, PD-1 and PD-L1 signaling, or COX-2. These findings thus indicate that gr-MDSCs from TB hosts have the unanticipated ability to restrict immunosuppressive Tregs.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular/inmunología , Tolerancia Inmunológica/inmunología , Células Mieloides/inmunología , Neoplasias Experimentales/inmunología , Escape del Tumor/inmunología , Animales , Linfocitos T CD4-Positivos/citología , Comunicación Celular/inmunología , Separación Celular , Femenino , Citometría de Flujo , Factores de Transcripción Forkhead/inmunología , Inmunohistoquímica , Subunidad alfa del Receptor de Interleucina-2/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Na+/H+ exchanger 3 (NHE3) provides a major route for intestinal Na+ absorption. NHE3 has been considered a target of proinflammatory cytokines and enteropathogenic bacteria, and impaired NHE3 expression and/or activity may be responsible for inflammation-associated diarrhea. However, the possibility of loss of NHE3 function reciprocally affecting gut immune homeostasis has not been investigated. In this report, we describe that NHE3-deficient mice spontaneously develop colitis restricted to distal colonic mucosa. NHE3(-/-) mice housed in a conventional facility exhibited phenotypic features such as mild diarrhea, occasional rectal prolapse, and reduced body weight. Genomewide microarray analysis identified not only a large group of transport genes that potentially represent an adaptive response, but also a considerable number of genes consistent with an inflammatory response. Histological examination demonstrated changes in the distal colon consistent with active inflammation, including crypt hyperplasia with an increased number of 5-bromo-2'-deoxyuridine-positive cells, diffuse neutrophilic infiltrate with concomitant 15-fold increase in matrix metalloproteinase 8 expression, an increased number of pSer276-RelA-positive cells, and a significant decrease in periodic acid-Schiff-positive goblet cells. Real-time PCR demonstrated elevated expression of inducible nitric oxide synthase (38-fold), TNF-alpha (6-fold), macrophage inflammatory protein-2 (48-fold), and IL-18 (3-fold) in the distal colon of NHE3(-/-) mice. NHE3(-/-) mice showed enhanced bacterial adhesion and translocation in the distal colon. Colitis was ameliorated by oral administration of broad-spectrum antibiotics. In conclusion, NHE3 deficiency leads to an exacerbated innate immune response, an observation suggesting a potentially novel role of NHE3 as a modifier gene, which when downregulated during infectious or chronic colitis may modulate the extent and severity of colonic inflammation.
Asunto(s)
Colitis/metabolismo , Perfilación de la Expresión Génica , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Antiinfecciosos/uso terapéutico , Ciprofloxacina/uso terapéutico , Colitis/tratamiento farmacológico , Colon/patología , Eliminación de Gen , Regulación de la Expresión Génica/fisiología , Metronidazol/uso terapéutico , Ratones , Intercambiador 3 de Sodio-Hidrógeno , Aumento de PesoRESUMEN
Sodium butyrate (NaB) stimulates sodium and water absorption by inducing colonic Na(+)/H(+) exchange. NaB induces Na(+)/H(+) exchanger (NHE)3 activity and protein and mRNA expression both in vivo and in vitro. Our previously published observations indicated that this induction is Ser/Thr kinase dependent and that NaB-responsive elements were localized within -320/-34 bp of the rat NHE3 promoter. Here we further delineate the mechanism of NaB-mediated NHE3 gene transcription. Transient and stable transfection of Caco-2 cells with NHE3 gene reporter constructs identified Sp binding site SpB at position -58/-55 nt as critical for NaB-mediated induction. Gel mobility shift (GMSA) and DNA affinity precipitation assays indicated NaB-induced binding of Sp3 and decreased binding of Sp1 to SpB element. While no changes in expression of Sp1 or Sp3 were noted, NaB induced phosphorylation of Sp1 and acetylation of Sp3. Sp3 was a more potent inducer of NHE3 gene transcription, which suggested that change in balance, favoring binding of Sp3 to the SpB site, would result in significant increase in NHE3 promoter activity. Small interfering RNA studies in Caco-2 cells and data from NaB-treated SL2 cells used as a reconstitution model confirmed this hypothesis. In addition to the SpB site, which played a permissive role, an upstream novel butyrate response element located at -196/-175 nt was necessary for maximal induction. GMSA identified a protein-DNA complex with a -196/-175 nt probe; this interaction was not affected by NaB treatment, thus suggesting that in response to NaB Sp3 binding to site SpB precedes and results in recruitment of the putative factor to this upstream site.
Asunto(s)
Antidiarreicos/farmacología , Ácido Butírico/farmacología , Mucosa Intestinal/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de los fármacos , Intercambiadores de Sodio-Hidrógeno/metabolismo , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp3/metabolismo , Transcripción Genética/efectos de los fármacos , Acetilación , Animales , Células CACO-2 , ADN/metabolismo , Elementos de Facilitación Genéticos/efectos de los fármacos , Genes Reporteros , Humanos , Mucosa Intestinal/metabolismo , Luciferasas de Renilla , Fosforilación , Procesamiento Proteico-Postraduccional/efectos de los fármacos , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/genética , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp3/genética , Activación Transcripcional/efectos de los fármacos , Transfección , Regulación hacia ArribaRESUMEN
In some carcinomas such as digestive tract carcinomas, bone marrow infiltration by tumor cells is a frequent event but usually remains a micrometastatic disease and rarely induces overt bone lesions. The mechanisms responsible for the control of these metastases in the bone marrow remain poorly known. We show that freshly isolated bone marrow cells from human, murine and rat origin rapidly kill a wide range of syngeneic or xenogeneic carcinoma cell lines in culture. Further analysis of this cytotoxic process in the rat indicated that neither resident bone marrow macrophages nor NK cells were responsible for this cytotoxic effect that was restricted to a subpopulation of bone marrow cells expressing CD90 (Thy-1), a marker of hemopoietic precursors. The tumoricidal activity of these cells did not require long-term culture nor addition of exogenous cytokines or growth factors. A subset of CD90+ cells that rapidly differentiates into CD163(ED2)-expressing macrophages was observed to be responsible for tumor cell killing. These macrophages induced a non-apoptotic death of tumor cells, a process that required both a direct interaction with the tumor cell and nitric oxide (NO) production through the activation of inducible nitric oxide-synthase (iNOS). This ability of pluripotent hemopoietic stem cells to rapidly differentiate into macrophages capable of killing invasive tumor cells may account for the limited expansion of micrometastases of some carcinomas in the bone marrow.