RESUMO
In this study, we aimed to apply the cytokine IL-36γ to cancer immunotherapy by constructing new oncolytic vaccinia viruses (OV) expressing interleukin-36γ (IL-36γ-OVs), leveraging unique synergism between OV and IL-36γ's ability to promote antitumor adaptive immunity and modulate tumor microenvironment (TME). IL-36γ-OV had dramatic therapeutic efficacies in multiple murine tumor models, frequently leading to complete cancer eradication in large fractions of mice. Mechanistically, IL-36-γ-armed OV induced infiltration of lymphocytes and dendritic cells, decreased myeloid-derived suppressor cells and M2-like tumor-associated macrophages, and T cell differentiation into effector cells. Further study showed that IL-36γ-OV increased the number of tumor antigen-specific CD4+ and CD8+ T cells and the therapeutic efficacy depended on both CD8+ and CD4+ T cells. These results demonstrate that these IL36γ-armed OVs exert potent therapeutic efficacy mainly though antitumor immunity and they may hold great potential to advance treatment in human cancer patients.
Assuntos
Imunidade Adaptativa , Terapia Genética , Vetores Genéticos/genética , Interleucina-1/genética , Neoplasias/imunologia , Neoplasias/terapia , Terapia Viral Oncolítica , Vírus Oncolíticos/genética , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Citotoxicidade Imunológica , Modelos Animais de Doenças , Expressão Gênica , Engenharia Genética , Vetores Genéticos/administração & dosagem , Humanos , Melanoma Experimental , Camundongos , Imagem Molecular , Neoplasias/diagnóstico , Neoplasias/genética , Terapia Viral Oncolítica/efeitos adversos , Terapia Viral Oncolítica/métodos , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Enteroviruses are among the most common viral infectious agents of humans and are primarily transmitted by the fecal-oral route. However, the events associated with enterovirus infections of the human gastrointestinal tract remain largely unknown. Here, we used stem cell-derived enteroids from human small intestines to study enterovirus infections of the intestinal epithelium. We found that enteroids were susceptible to infection by diverse enteroviruses, including echovirus 11 (E11), coxsackievirus B (CVB), and enterovirus 71 (EV71), and that contrary to an immortalized intestinal cell line, enteroids induced antiviral and inflammatory signaling pathways in response to infection in a virus-specific manner. Furthermore, using the Notch inhibitor dibenzazepine (DBZ) to drive cellular differentiation into secretory cell lineages, we show that although goblet cells resist E11 infection, enteroendocrine cells are permissive, suggesting that enteroviruses infect specific cell populations in the human intestine. Taken together, our studies provide insights into enterovirus infections of the human intestine, which could lead to the identification of novel therapeutic targets and/or strategies to prevent or treat infections by these highly clinically relevant viruses.
Assuntos
Infecções por Enterovirus/virologia , Enterovirus/fisiologia , Intestino Delgado/virologia , Organoides/virologia , Células CACO-2 , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Células Cultivadas , Dibenzazepinas/farmacologia , Resistência à Doença/genética , Infecções por Enterovirus/metabolismo , Inibidores Enzimáticos/farmacologia , Perfilação da Expressão Gênica/métodos , Interações Hospedeiro-Patógeno , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/virologia , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Organoides/citologia , Organoides/metabolismo , Transdução de Sinais/genéticaRESUMO
Oncolytic immunotherapy is a promising novel therapeutic for cancer, and further preclinical studies may maximize its therapeutic efficacy. In this study, we construct a novel oncolytic vaccinia virus (VV) expressing a superagoinst IL-15, a fusion protein of IL-15 and IL-15Ralpha. This virus, named vvDD-IL15-Rα, possesses similar replication efficiency as the parental virus vvDD yet leads to significantly more regression of the disease and extends the survival of mice bearing MC38 colon or ID8 ovarian cancer. This novel virus elicits potent adaptive antitumor immunity as shown by ELISPOT assays for interferon-gamma-secreting CD8+ T cells and by the rejection of tumor implants upon re-challenge in the mice, which were previously cured by vvDD-IL15-Rα treatment. In vivo cell depletion assays with antibodies showed that this antitumor activity is highly dependent on CD8+ T cells but much less so on CD4+ T cells and NK cells. Finally, the combination of the oncolytic immunotherapy with anti-PD-1 antibody dramatically improves the therapeutic outcome compared to either anti-PD-1 alone or vvDD-IL15-Rα alone. These results demonstrate that the IL-15-IL-15Rα fusion protein-expressing OV elicits potent antitumor immunity, and rational combination with PD-1 blockade leads to dramatic tumor regression and prolongs the survival of mice bearing colon or ovarian cancers.
Assuntos
Subunidade alfa de Receptor de Interleucina-15/genética , Interleucina-15/genética , Neoplasias/terapia , Receptor de Morte Celular Programada 1/genética , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Terapia Combinada , Feminino , Humanos , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/imunologia , Imunoterapia/métodos , Interferon gama/genética , Interleucina-15/administração & dosagem , Subunidade alfa de Receptor de Interleucina-15/administração & dosagem , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Camundongos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Terapia Viral Oncolítica/métodos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Necrotising enterocolitis (NEC) is a common disease in premature infants characterised by intestinal ischaemia and necrosis. The only effective preventative strategy against NEC is the administration of breast milk, although the protective mechanisms remain unknown. We hypothesise that an abundant human milk oligosaccharide (HMO) in breast milk, 2'-fucosyllactose (2'FL), protects against NEC by enhancing intestinal mucosal blood flow, and we sought to determine the mechanisms underlying this protection. Administration of HMO-2'FL protected against NEC in neonatal wild-type mice, resulted in a decrease in pro-inflammatory markers and preserved the small intestinal mucosal architecture. These protective effects occurred via restoration of intestinal perfusion through up-regulation of the vasodilatory molecule endothelial nitric oxide synthase (eNOS), as administration of HMO-2'FL to eNOS-deficient mice or to mice that received eNOS inhibitors did not protect against NEC, and by 16S analysis HMO-2'FL affected the microbiota of the neonatal mouse gut, although these changes do not seem to be the primary mechanism of protection. Induction of eNOS by HMO-2'FL was also observed in cultured endothelial cells, providing a link between eNOS and HMO in the endothelium. These data demonstrate that HMO-2'FL protects against NEC in part through maintaining mesenteric perfusion via increased eNOS expression, and suggest that the 2'FL found in human milk may be mediating some of the protective benefits of breast milk in the clinical setting against NEC.
Assuntos
Enterocolite Necrosante/prevenção & controle , Doenças do Prematuro/fisiopatologia , Leite Humano/química , Circulação Esplâncnica/efeitos dos fármacos , Trissacarídeos/administração & dosagem , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Enterocolite Necrosante/fisiopatologia , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Recém-Nascido , Mucosa Intestinal/irrigação sanguínea , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microbiota/efeitos dos fármacos , Óxido Nítrico/análise , Óxido Nítrico Sintase Tipo III/deficiência , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/fisiologiaRESUMO
Necrotizing enterocolitis (NEC) is a devastating disease of premature infants characterized by severe intestinal necrosis and for which breast milk represents the most effective protective strategy. Previous studies have revealed a critical role for the lipopolysaccharide receptor toll-like receptor 4 (TLR4) in NEC development through its induction of mucosal injury, yet the reasons for which intestinal ischemia in NEC occurs in the first place remain unknown. We hypothesize that TLR4 signaling within the endothelium plays an essential role in NEC development by regulating perfusion to the small intestine via the vasodilatory molecule endothelial nitric oxide synthase (eNOS). Using a unique mouse system in which we selectively deleted TLR4 from the endothelium, we now show that endothelial TLR4 activation is required for NEC development and that endothelial TLR4 activation impairs intestinal perfusion without effects on other organs and reduces eNOS expression via activation of myeloid differentiation primary response gene 88. NEC severity was significantly increased in eNOS(-/-) mice and decreased upon administration of the phosphodiesterase inhibitor sildenafil, which augments eNOS function. Strikingly, compared with formula, human and mouse breast milk were enriched in sodium nitrate--a precursor for enteral generation of nitrite and nitric oxide--and repletion of formula with sodium nitrate/nitrite restored intestinal perfusion, reversed the deleterious effects of endothelial TLR4 signaling, and reduced NEC severity. These data identify that endothelial TLR4 critically regulates intestinal perfusion leading to NEC and reveal that the protective properties of breast milk involve enhanced intestinal microcirculatory integrity via augmentation of nitrate-nitrite-NO signaling.
Assuntos
Enterocolite Necrosante/etiologia , Mucosa Intestinal/irrigação sanguínea , Microcirculação/fisiologia , Transdução de Sinais/fisiologia , Receptor 4 Toll-Like/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Enterocolite Necrosante/tratamento farmacológico , Enterocolite Necrosante/metabolismo , Fórmulas Infantis/química , Fórmulas Infantis/farmacologia , Camundongos , Camundongos Knockout , Microcirculação/efeitos dos fármacos , Microscopia Confocal , Leite Humano/química , Nitratos/análise , Nitratos/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Nitritos/metabolismo , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Purinas/farmacologia , Purinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Citrato de Sildenafila , Sulfonas/farmacologia , Sulfonas/uso terapêutico , Receptor 4 Toll-Like/deficiênciaRESUMO
The cellular cues that regulate the apoptosis of intestinal stem cells (ISCs) remain incompletely understood, yet may play a role in diseases characterized by ISC loss including necrotizing enterocolitis (NEC). Toll-like receptor-4 (TLR4) was recently found to be expressed on ISCs, where its activation leads to ISC apoptosis through mechanisms that remain incompletely explained. We now hypothesize that TLR4 induces endoplasmic reticulum (ER) stress within ISCs, leading to their apoptosis in NEC pathogenesis, and that high ER stress within the premature intestine predisposes to NEC development. Using transgenic mice and cultured enteroids, we now demonstrate that TLR4 induces ER stress within Lgr5 (leucine-rich repeat-containing G-protein-coupled receptor 5)-positive ISCs, resulting in crypt apoptosis. TLR4 signaling within crypts was required, because crypt ER stress and apoptosis occurred in TLR4(ΔIEC-OVER) mice expressing TLR4 only within intestinal crypts and epithelium, but not TLR4(ΔIEC) mice lacking intestinal TLR4. TLR4-mediated ER stress and apoptosis of ISCs required PERK (protein kinase-related PKR-like ER kinase), CHOP (C/EBP homologous protein), and MyD88 (myeloid differentiation primary response gene 88), but not ATF6 (activating transcription factor 6) or XBP1 (X-box-binding protein 1). Human and mouse NEC showed high crypt ER stress and apoptosis, whereas genetic inhibition of PERK or CHOP attenuated ER stress, crypt apoptosis, and NEC severity. Strikingly, using intragastric delivery into fetal mouse intestine, prevention of ER stress reduced TLR4-mediated ISC apoptosis and mucosal disruption. These findings identify a novel link between TLR4-induced ER stress and ISC apoptosis in NEC pathogenesis and suggest that increased ER stress within the premature bowel predisposes to NEC development.
Assuntos
Estresse do Retículo Endoplasmático , Enterocolite Necrosante/metabolismo , Mucosa Intestinal/metabolismo , Células-Tronco/metabolismo , Receptor 4 Toll-Like/metabolismo , Fator 6 Ativador da Transcrição/genética , Fator 6 Ativador da Transcrição/metabolismo , Animais , Apoptose/genética , Enterocolite Necrosante/genética , Enterocolite Necrosante/patologia , Células HEK293 , Humanos , Mucosa Intestinal/patologia , Camundongos , Camundongos Knockout , Células-Tronco/patologia , Receptor 4 Toll-Like/genética , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
Necrotizing enterocolitis (NEC) develops in response to elevated TLR4 signaling in the newborn intestinal epithelium and is characterized by TLR4-mediated inhibition of enterocyte migration and reduced mucosal healing. The downstream processes by which TLR4 impairs mucosal healing remain incompletely understood. In other systems, TLR4 induces autophagy, an adaptive response to cellular stress. We now hypothesize that TLR4 induces autophagy in enterocytes and that TLR4-induced autophagy plays a critical role in NEC development. Using mice selectively lacking TLR4 in enterocytes (TLR4(ΔIEC)) and in TLR4-deficient cultured enterocytes, we now show that TLR4 activation induces autophagy in enterocytes. Immature mouse and human intestine showed increased expression of autophagy genes compared with full-term controls, and NEC development in both mouse and human was associated with increased enterocyte autophagy. Importantly, using mice in which we selectively deleted the autophagy gene ATG7 from the intestinal epithelium (ATG7(ΔIEC)), the induction of autophagy was determined to be required for and not merely a consequence of NEC, because ATG7(ΔIEC) mice were protected from NEC development. In defining the mechanisms involved, TLR4-induced autophagy led to impaired enterocyte migration both in vitro and in vivo, which in cultured enterocytes required the induction of RhoA-mediated stress fibers. These findings depart from current dogma in the field by identifying a unique effect of TLR4-induced autophagy within the intestinal epithelium in the pathogenesis of NEC and identify that the negative consequences of autophagy on enterocyte migration play an essential role in its development.
Assuntos
Autofagia , Movimento Celular , Enterocolite Necrosante/etiologia , Enterócitos/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Autofagia/genética , Linhagem Celular , Movimento Celular/genética , Modelos Animais de Doenças , Enterocolite Necrosante/patologia , Humanos , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Transgênicos , Receptor 4 Toll-Like/genética , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
The fetal intestinal mucosa is characterized by elevated Toll-like receptor 4 (TLR4) expression, which can lead to the development of necrotizing enterocolitis (NEC)--a devastating inflammatory disease of the premature intestine--upon exposure to microbes. To define endogenous strategies that could reduce TLR4 signaling, we hypothesized that amniotic fluid can inhibit TLR4 signaling within the fetal intestine and attenuate experimental NEC, and we sought to determine the mechanisms involved. We show here that microinjection of amniotic fluid into the fetal (embryonic day 18.5) gastrointestinal tract reduced LPS-mediated signaling within the fetal intestinal mucosa. Amniotic fluid is abundant in EGF, which we show is required for its inhibitory effects on TLR4 signaling via peroxisome proliferator-activated receptor, because inhibition of EGF receptor (EGFR) with cetuximab or EGF-depleted amniotic fluid blocked the inhibitory effects of amniotic fluid on TLR4, whereas amniotic fluid did not prevent TLR4 signaling in EGFR- or peroxisome proliferator-activated receptor γ-deficient enterocytes or in mice deficient in intestinal epithelial EGFR, and purified EGF attenuated the exaggerated intestinal mucosal TLR4 signaling in wild-type mice. Moreover, amniotic fluid-mediated TLR4 inhibition reduced the severity of NEC in mice through EGFR activation. Strikingly, NEC development in both mice and humans was associated with reduced EGFR expression that was restored upon the administration of amniotic fluid in mice or recovery from NEC in humans, suggesting that a lack of amniotic fluid-mediated EGFR signaling could predispose to NEC. These findings may explain the unique susceptibility of premature infants to the development of NEC and offer therapeutic approaches to this devastating disease.
Assuntos
Líquido Amniótico/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mucosa Intestinal/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Linhagem Celular , Enterocolite Necrosante/metabolismo , Enterócitos/metabolismo , Receptores ErbB/metabolismo , Humanos , Recém-Nascido , Mucosa Intestinal/embriologia , Intestinos/embriologia , Camundongos , Microscopia Confocal/métodos , Transdução de Sinais , Fatores de TempoRESUMO
Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal-related mortality in premature infants, and it develops under conditions of exaggerated TLR4 signaling in the newborn intestinal epithelium. Because NEC does not develop spontaneously, despite the presence of seemingly tonic stimulation of intestinal TLR4, we hypothesized that mechanisms must exist to constrain TLR4 signaling that become diminished during NEC pathogenesis and focused on the intracellular stress response protein and chaperone heat shock protein-70 (Hsp70). We demonstrate that the induction of intracellular Hsp70 in enterocytes dramatically reduced TLR4 signaling, as assessed by LPS-induced NF-κB translocation, cytokine expression, and apoptosis. These findings were confirmed in vivo, using mice that either globally lacked Hsp70 or overexpressed Hsp70 within the intestinal epithelium. TLR4 activation itself significantly increased Hsp70 expression in enterocytes, which provided a mechanism of autoinhibition of TLR4 signaling in enterocytes. In seeking to define the mechanisms involved, intracellular Hsp70-mediated inhibition of TLR4 signaling required both its substrate-binding EEVD domain and association with the cochaperone CHIP, resulting in ubiquitination and proteasomal degradation of TLR4. The expression of Hsp70 in the intestinal epithelium was significantly decreased in murine and human NEC compared with healthy controls, suggesting that loss of Hsp70 protection from TLR4 could lead to NEC. In support of this, intestinal Hsp70 overexpression in mice and pharmacologic upregulation of Hsp70 reversed TLR4-induced cytokines and enterocyte apoptosis, as well as prevented and treated experimental NEC. Thus, a novel TLR4 regulatory pathway exists within the newborn gut involving Hsp70 that may be pharmacologically activated to limit NEC severity.
Assuntos
Proteínas de Choque Térmico HSP70/imunologia , Mucosa Intestinal/imunologia , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Enterocolite Necrosante/imunologia , Enterocolite Necrosante/metabolismo , Enterocolite Necrosante/patologia , Feminino , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Recém-Nascido , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , NF-kappa B/imunologia , NF-kappa B/metabolismo , Complexo de Endopeptidases do Proteassoma/imunologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Estrutura Terciária de Proteína , Proteólise/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Ubiquitina-Proteína Ligases/imunologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/imunologiaRESUMO
Factors regulating the proliferation and apoptosis of intestinal stem cells (ISCs) remain incompletely understood. Because ISCs exist among microbial ligands, immune receptors such as toll-like receptor 4 (TLR4) could play a role. We now hypothesize that ISCs express TLR4 and that the activation of TLR4 directly on the intestinal stem cells regulates their ability to proliferate or to undergo apoptosis. Using flow cytometry and fluorescent in situ hybridization for the intestinal stem cell marker Lgr5, we demonstrate that TLR4 is expressed on the Lgr5-positive intestinal stem cells. TLR4 activation reduced proliferation and increased apoptosis in ISCs both in vivo and in ISC organoids, a finding not observed in mice lacking TLR4 in the Lgr5-positive ISCs, confirming the in vivo significance of this effect. To define molecular mechanisms involved, TLR4 inhibited ISC proliferation and increased apoptosis via the p53-up-regulated modulator of apoptosis (PUMA), as TLR4 did not affect crypt proliferation or apoptosis in organoids or mice lacking PUMA. In vivo effects of TLR4 on ISCs required TIR-domain-containing adapter-inducing interferon-ß (TRIF) but were independent of myeloid-differentiation primary response-gene 88 (MYD88) and TNFα. Physiological relevance was suggested, as TLR4 activation in necrotizing enterocolitis led to reduced proliferation and increased apoptosis of the intestinal crypts in a manner that could be reversed by inhibition of PUMA, both globally or restricted to the intestinal epithelium. These findings illustrate that TLR4 is expressed on ISCs where it regulates their proliferation and apoptosis through activation of PUMA and that TLR4 regulation of ISCs contributes to the pathogenesis of necrotizing enterocolitis.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Proliferação de Células , Mucosa Intestinal/patologia , Células-Tronco/metabolismo , Receptor 4 Toll-Like/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/fisiologia , Enterocolite Necrosante/metabolismo , Enterocolite Necrosante/patologia , Técnicas de Inativação de Genes , Íleo/patologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/metabolismo , Ratos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais , Células-Tronco/imunologia , Células-Tronco/fisiologia , Receptor 4 Toll-Like/genética , Ativação Transcricional , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/fisiologiaRESUMO
BACKGROUND & AIMS: Little is known about factors that regulate intestinal epithelial differentiation; microbial recognition receptors such as Toll-like receptor (TLR)4 might be involved. We investigated whether intestinal TLR4 regulates epithelial differentiation and is involved in development of necrotizing enterocolitis (NEC) of the immature intestine. METHODS: Mice with conditional disruption of TLR4 in the intestinal epithelium and TLR4 knockout (TLR4(-/-)) mice were generated by breeding TLR4(loxp/loxp) mice with villin-cre and Ella-cre, respectively. Enterocytes that did not express or overexpressed TLR4 were created by lentiviral or adenoviral transduction. Intestinal organoids were cultured on tissue matrices. Bile acids were measured by colorimetric assays, and microbial composition was determined by 16S pyrosequencing. NEC was induced in 7- to 10-day-old mice by induction of hypoxia twice daily for 4 days. RESULTS: TLR4(-/-) mice and mice with enterocyte-specific deletion of TLR4 were protected from NEC; epithelial differentiation into goblet cells was increased via suppressed Notch signaling in the small intestinal epithelium. TLR4 also regulates differentiation of goblet cells in intestinal organoid and enterocyte cell cultures; differentiation was increased on deletion of TLR4 and restored when TLR4 was expressed ectopically. TLR4 signaling via Notch was increased in intestinal tissue samples from patients with NEC, and numbers of goblet cells were reduced. 16S pyrosequencing revealed that wild-type and TLR4-deficient mice had similar microbial profiles; increased numbers of goblet cells were observed in mice given antibiotics. TLR4 deficiency reduced levels of luminal bile acids in vivo, and addition of bile acids to TLR4-deficient cell cultures prevented differentiation of goblet cells. CONCLUSIONS: TLR4 signaling and Notch are increased in intestinal tissues of patients with NEC and required for induction of NEC in mice. TLR4 prevents goblet cell differentiation, independently of the microbiota. Bile acids might initiate goblet cell development.
Assuntos
Diferenciação Celular , Enterocolite Necrosante/metabolismo , Células Caliciformes/metabolismo , Intestino Delgado/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Animais Recém-Nascidos , Ácidos e Sais Biliares/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Enterocolite Necrosante/etiologia , Enterocolite Necrosante/genética , Enterocolite Necrosante/microbiologia , Enterocolite Necrosante/patologia , Enterocolite Necrosante/prevenção & controle , Células Caliciformes/microbiologia , Células Caliciformes/patologia , Humanos , Hipóxia/complicações , Fórmulas Infantis , Recém-Nascido , Intestino Delgado/microbiologia , Intestino Delgado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Organoides , Interferência de RNA , Ratos , Receptores Notch/metabolismo , Transdução de Sinais , Técnicas de Cultura de Tecidos , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genética , TransfecçãoRESUMO
Ferroptosis is a type of programmed cell death dependent on iron and characterized by the accumulation of lipid peroxides. In this study, we explore the combination of a ferroptosis activator with an oncolytic vaccinia virus in tumor models. Erastin induced cell death in hepatoma, colon, and ovarian cancer cells, but not in melanoma cancer cells. Erastin, not the oncolytic vaccinia virus (OVV), induced the expression of key marker genes for ferroptosis in cancer cells. In hepatocellular carcinoma and colon cancer models, either erastin or OVV inhibited tumor growth, but a combination of the two yielded the best therapeutic effects, as indicated by inhibited tumor growth or regression and longer host survival. Immunological analyses indicate that erastin alone had little or no effect on systemic immunity or local immunity in the tumor. However, when combined with OV, erastin enhanced the number of activated dendritic cells and the activity of tumor-infiltrating T lymphocytes as indicated by an increase in IFN-γ+CD8+ and PD-1+CD8+ T cells. These results demonstrate that erastin can exert cytotoxicity on cancer cells via ferroptosis, but has little effect on immune activity by itself. However, when combined with an OVV, erastin promoted antitumoral immunity and efficacy by increasing the number of activated dendritic cells and promoting the activities of tumor specific CD8+ T cells in the tumor.
RESUMO
Toll-like receptor-4 (TLR4) is the receptor for bacterial lipopolysaccharide, yet it may also respond to a variety of endogenous molecules. Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in newborn infants and is characterized by intestinal mucosal destruction and impaired enterocyte migration due to increased TLR4 signaling on enterocytes. The endogenous ligands for TLR4 that lead to impaired enterocyte migration remain unknown. High mobility group box-1 (HMGB1) is a DNA-binding protein that is released from injured cells during inflammation. We thus hypothesize that extracellular HMGB1 inhibits enterocyte migration via activation of TLR4 and sought to define the pathways involved. We now demonstrate that murine and human NEC are associated with increased intestinal HMGB1 expression, that serum HMGB1 is increased in murine NEC, and that HMGB1 inhibits enterocyte migration in vitro and in vivo in a TLR4-dependent manner. This finding was unique to enterocytes as HMGB1 enhanced migration of inflammatory cells in vitro and in vivo. In seeking to understand the mechanisms involved, TLR4-dependent HMGB1 signaling increased RhoA activation in enterocytes, increased phosphorylation of focal adhesion kinase, and increased phosphorylation of cofilin, resulting in increased stress fibers and focal adhesions. Using single cell force traction microscopy, the net effect of HMGB1 signaling was a TLR4-dependent increase in cell force adhesion, accounting for the impaired enterocyte migration. These findings demonstrate a novel pathway by which TLR4 activation by HMGB1 delays mucosal repair and suggest a novel potential therapeutic target in the amelioration of intestinal inflammatory diseases like NEC.
Assuntos
Movimento Celular/efeitos dos fármacos , Enterócitos/citologia , Proteína HMGB1/metabolismo , Proteína HMGB1/farmacologia , Mucosa Intestinal/metabolismo , Receptor 4 Toll-Like/metabolismo , Actinas/metabolismo , Animais , Linhagem Celular , Movimento Celular/genética , Quimiotaxia/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Enterocolite Necrosante/metabolismo , Enterócitos/efeitos dos fármacos , Citometria de Fluxo , Humanos , Técnicas In Vitro , Recém-Nascido , Mucosa Intestinal/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Camundongos , Receptor 4 Toll-Like/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
BACKGROUND & AIMS: Factors that regulate enterocyte apoptosis in necrotizing enterocolitis (NEC) remain incompletely understood, although Toll-like receptor-4 (TLR4) signaling in enterocytes plays a major role. Nucleotide-binding oligomerization domain-2 (NOD2) is an immune receptor that regulates other branches of the immune system, although its effects on TLR4 in enterocytes and its role in NEC remain unknown. We now hypothesize that activation of NOD2 in the newborn intestine inhibits TLR4, and that failure of NOD2 signaling leads to NEC through increased TLR4-mediated enterocyte apoptosis. METHODS: The effects of NOD2 on enterocyte TLR4 signaling and intestinal injury and repair were assessed in enterocytes lacking TLR4 or NOD2, in mice with intestinal-specific wild-type or dominant-negative TLR4 or NOD2, and in mice with NEC. A protein array was performed on NOD2-activated enterocytes to identify novel effector molecules involved. RESULTS: TLR4 activation caused apoptosis in newborn but not adult small intestine or colon, and its intestinal expression was influenced by NOD2. NOD2 activation inhibited TLR4 in enterocytes, but not macrophages, and reversed the effects of TLR4 on intestinal mucosal injury and repair. Protection from TLR4-induced enterocyte apoptosis by NOD2 required a novel pathway linking NOD2 with the apoptosis mediator second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low PI (SMAC-DIABLO), both in vitro and in vivo. Strikingly, activation of NOD2 reduced SMAC-DIABLO expression, attenuated the extent of enterocyte apoptosis, and reduced the severity of NEC. CONCLUSIONS: These findings reveal a novel inhibitory interaction between TLR4 and NOD2 signaling in enterocytes leading to the regulation of enterocyte apoptosis and suggest a therapeutic role for NOD2 in the protection of intestinal diseases such as NEC.
Assuntos
Endotoxemia/metabolismo , Enterocolite Necrosante/metabolismo , Mucosa Intestinal/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Fatores Etários , Animais , Animais Recém-Nascidos , Apoptose , Proteínas Reguladoras de Apoptose , Proteínas de Transporte/metabolismo , Linhagem Celular , Movimento Celular , Modelos Animais de Doenças , Endotoxemia/genética , Endotoxemia/patologia , Enterocolite Necrosante/genética , Enterocolite Necrosante/patologia , Enterocolite Necrosante/prevenção & controle , Enterócitos/metabolismo , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mitocondriais/metabolismo , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD2/agonistas , Proteína Adaptadora de Sinalização NOD2/deficiência , Proteína Adaptadora de Sinalização NOD2/genética , Análise Serial de Proteínas , Ratos , Índice de Gravidade de Doença , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/genética , Transdução GenéticaRESUMO
BACKGROUND & AIMS: Necrotizing enterocolitis (NEC), the leading cause of gastrointestinal death from gastrointestinal disease in preterm infants, is characterized by exaggerated TLR4 signaling and decreased enterocyte proliferation through unknown mechanisms. Given the importance of beta-catenin in regulating proliferation of many cell types, we hypothesize that TLR4 impairs enterocyte proliferation in NEC via impaired beta-catenin signaling. METHODS: Enterocyte proliferation was detected in IEC-6 cells or in ileum or colon from wild-type, TLR4-mutant, or TLR4(-/-) mice after induction of NEC or endotoxemia. beta-Catenin signaling was assessed by cell fractionation or immunoconfocal microscopy to detect its nuclear translocation. Activation and inhibition of beta-catenin were achieved via cDNA or small interfering RNA, respectively. TLR4 in the intestinal mucosa was inhibited with adenoviruses expressing dominant-negative TLR4. RESULTS: TLR4 activation significantly impaired enterocyte proliferation in the ileum but not colon in newborn but not adult mice and in IEC-6 enterocytes. beta-Catenin activation reversed these effects in vitro. To determine the mechanisms involved, TLR4 activation phosphorylated the upstream inhibitory kinase GSK3beta, causing beta-catenin degradation. NEC in both mouse and humans was associated with decreased beta-catenin and increased mucosal GSK3beta expression. Strikingly, the inhibition of enterocyte beta-catenin signaling in NEC could be reversed, and enterocyte proliferation restored, through adenoviral-mediated inhibition of TLR4 signaling in the small intestinal mucosa. CONCLUSION: We now report a novel pathway linking TLR4 with inhibition of beta-catenin signaling via GSK3beta activation, leading to reduced enterocyte proliferation in vitro and in vivo. These data provide additional insights into the pathogenesis of diseases of intestinal inflammation such as NEC.
Assuntos
Enterocolite Necrosante/metabolismo , Enterócitos/citologia , Enterócitos/metabolismo , Receptor 4 Toll-Like/metabolismo , beta Catenina/metabolismo , Adenoviridae/genética , Animais , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Células Cultivadas , Colo/patologia , Enterocolite Necrosante/patologia , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Íleo/patologia , Recém-Nascido , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Mutantes , Transdução de Sinais/fisiologia , Receptor 4 Toll-Like/genéticaRESUMO
Adoptive cell therapy (ACT) using autologous tumor infiltrating lymphocytes (TIL) achieves durable clinical benefit for patients from whom these cells can be derived in advanced metastatic melanoma but is limited in most solid tumors as a result of immune escape and exclusion. A tumor microenvironment (TME) priming strategy to improve the quantity and quality of TIL represents an important tactic to explore. Oncolytic viruses expressing immune stimulatory cytokines induce a potent inflammatory response that may enhance infiltration and activation of T cells. In this study, we examined the ability of an attenuated oncolytic vaccinia virus expressing IL15/IL15Rα (vvDD-IL15/Rα) to enhance recovery of lavage T cells in peritoneal carcinomatosis (PC). We found that intraperitoneal (IP) vvDD-IL15/Rα treatment of animals bearing PC resulted in a significant increase in cytotoxic function and memory formation in CD8+ T cells in peritoneal fluid. Using tetramers for vaccinia virus B8R antigen and tumor rejection antigen p15E, we found that the expanded population of peritoneal CD8+ T cells are specific for vaccinia or tumor with increased tumor-specificity over time, reinforced with viral clearance. Application of these vvDD-IL15/Rα induced CD8+ T cells in ACT of a lethal model of PC significantly increased survival. In addition, we found in patients with peritoneal metastases from various primary solid tumors that peritoneal T cells could be recovered but were exhausted with infrequent tumor-reactivity. If clinically translatable, vvDD-IL15/Rα in vivo priming would greatly expand the number of patients with advanced metastatic cancers responsive to T cell therapy.
Assuntos
Vetores Genéticos/efeitos adversos , Imunoterapia Adotiva , Ativação Linfocitária/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Vírus Oncolíticos/imunologia , Neoplasias Peritoneais/terapia , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Vetores Genéticos/genética , Humanos , Imunofenotipagem , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Interleucina-15/metabolismo , Subunidade alfa de Receptor de Interleucina-15/metabolismo , Camundongos , Terapia Viral Oncolítica/efeitos adversos , Vírus Oncolíticos/genética , Neoplasias Peritoneais/secundário , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Background: Newly emerging cancer immunotherapy has led to significant progress in cancer treatment; however, its efficacy is limited in solid tumors since the majority of them are "cold" tumors. Oncolytic viruses, especially when properly armed, can directly target tumor cells and indirectly modulate the tumor microenvironment (TME), resulting in "hot" tumors. These viruses can be applied as a cancer immunotherapy approach either alone or in combination with other cancer immunotherapies. Cytokines are good candidates to arm oncolytic viruses. IL-23, an IL-12 cytokine family member, plays many roles in cancer immunity. Here, we used oncolytic vaccinia viruses to deliver IL-23 variants into the tumor bed and explored their activity in cancer treatment on multiple tumor models. Methods: Oncolytic vaccinia viruses expressing IL-23 variants were generated by homologue recombination. The characteristics of these viruses were in vitro evaluated by RT-qPCR, ELISA, flow cytometry and cytotoxicity assay. The antitumor effects of these viruses were evaluated on multiple tumor models in vivo and the mechanisms were investigated by RT-qPCR and flow cytometry. Results: IL-23 prolonged viral persistence, probably mediated by up-regulated IL-10. The sustainable IL-23 expression and viral oncolysis elevated the expression of Th1 chemokines and antitumor factors such as IFN-γ, TNF-α, Perforin, IL-2, Granzyme B and activated T cells in the TME, transforming the TME to be more conducive to antitumor immunity. This leads to a systemic antitumor effect which is dependent on CD8+ and CD4+ T cells and IFN-γ. Oncolytic vaccinia viruses could not deliver stable IL-23A to the tumor, attributed to the elevated tristetraprolin which can destabilize the IL-23A mRNA after the viral treatment; whereas vaccinia viruses could deliver membrane-bound IL-23 to elicit a potent antitumor effect which might avoid the possible toxicity normally associated with systemic cytokine exposure. Conclusion: Either secreted or membrane-bound IL-23-armed vaccinia virus can induce potent antitumor effects and IL-23 is a candidate cytokine to arm oncolytic viruses for cancer immunotherapy.
Assuntos
Adenocarcinoma/terapia , Neoplasias do Colo/terapia , Imunoterapia/métodos , Interleucina-23/farmacologia , Vírus Oncolíticos/genética , Microambiente Tumoral/imunologia , Vaccinia virus/genética , Adenocarcinoma/imunologia , Adenocarcinoma/virologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Quimiocinas/metabolismo , Neoplasias do Colo/imunologia , Neoplasias do Colo/virologia , Modelos Animais de Doenças , Feminino , Vetores Genéticos , Granzimas/metabolismo , Humanos , Interferon gama/metabolismo , Interleucina-12/metabolismo , Interleucina-2/metabolismo , Interleucina-23/genética , Interleucina-23/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Vírus Oncolíticos/metabolismo , Perforina/metabolismo , Microambiente Tumoral/genética , Fator de Necrose Tumoral alfa/metabolismo , Vaccinia virus/metabolismoRESUMO
Adoptive cell therapy (ACT) using tumor-specific tumor-infiltrating lymphocytes (TILs) has demonstrated success in patients where tumor-antigen specific TILs can be harvested from the tumor, expanded, and re-infused in combination with a preparatory regimen and IL2. One major issue for non-immunogenic tumors has been that the isolated TILs lack tumor specificity and thus possess limited in vivo therapeutic function. An oncolytic virus (OV) mediates an immunogenic cell death for cancer cells, leading to elicitation and dramatic enhancement of tumor-specific TILs. We hypothesized that the tumor-specific TILs elicited and promoted by an OV would be a great source for ACT for solid cancer. In this study, we show that a local injection of oncolytic poxvirus in MC38 tumor with low immunogenicity in C57BL/6 mice, led to elicitation and accumulation of tumor-specific TILs in the tumor tissue. Our analyses indicated that IL2-armed OV-elicited TILs contain lower quantities of exhausted PD-1hiTim-3+ CD8+ T cells and regulatory T cells. The isolated TILs from IL2-expressing OV-treated tumor tissue retained high tumor specificity after expansion ex vivo. These TILs resulted in significant tumor regression and improved survival after adoptive transfer in mice with established MC38 tumor. Our study showcases the feasibility of using an OV to induce tumor-reactive TILs that can be expanded for ACT.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Imunoterapia Adotiva/métodos , Linfócitos do Interstício Tumoral/imunologia , Vírus Oncolíticos/imunologia , Animais , Feminino , Humanos , CamundongosRESUMO
Successful in situ therapeutic vaccination would allow locally delivered oncolytic virus (OV) to exert systemic immunologic effects on metastases and improve survival. We have utilized bilateral flank tumor models to determine the most efficacious regimens of in situ vaccination. Intratumoral injection with membrane-tethered interleukin -2-armed OV (vvDD-mIL2) plus a Toll-like receptor 9 ligand (CpG) yielded systemic immunization and decreased tumor growth in a contralateral, noninjected tumor. Our main aims were to study the tumor immune microenvironment (TME) after vaccination and identify additional immune adjuvants that may improve the systemic tumor-specific immunity. Immunological profiles in the spleen showed an increased CD8+ T cell/regulatory T cell (Treg) ratio and increased CD11c+ cells after dual injection in one flank tumor. Concurrently, there was increased infiltration of tumor necrosis factor alpha (TNF-α)+CD8+ T cells and interferon gamma (IFN-γ)+CD4+ T cells and reduced CTLA-4+PD-1+CD8+ T cells in the contralateral, noninjected tumor. The anti-tumoral activity depended on CD8+ T cells and IFN-γ, but not CD4+ T cells. Based on the negative immune components still existing in the untreated tumors, we investigated additional adjuvants: clodronate liposome-mediated depletion of macrophages plus anti-PD-1 therapy. This regimen dramatically reduced the tumor burden in the noninjected tumor and increased median survival by 87%, suggesting that inhibition/elimination of suppressive components in the tumor microenvironment (TME) can improve therapeutic outcomes. This study emphasizes the importance of immune profiling to design rational, combined immunotherapy regimens ultimately to impact patient survival.
RESUMO
Immune checkpoint blockade is arguably the most effective current cancer therapy approach; however, its efficacy is limited to patients with "hot" tumors, warranting an effective approach to transform "cold" tumors. Oncolytic viruses (especially properly armed ones) have positive effects on almost every aspect of the cancer-immunity cycle and can change the cancer-immune set point of a tumor. Here, we tested whether oncolytic vaccinia virus delivering tethered interleukin 12 (IL-12) could turn a "cold" tumor into a "hot" tumor while avoiding IL-12's systemic toxicity. Our data demonstrated that tethered IL-12 could be maintained in the tumor without treatment-induced toxic side effects. Moreover, the treatment facilitated tumor infiltration of more activated CD4+ and CD8+ T cells and less Tregs, granulocytic myeloid-derivedsuppressor cells, and exhausted CD8+ T cells, with increased interferon γ and decreased transforming growth factor ß, cyclooxygenase-2, and vascular endothelial growth factor expression, leading to transformed, immunogenic tumors and improved survival. Combined with programmed cell death 1 blockade, vaccinia virus expressing tethered IL-12 cured all mice with late-stage colon cancer, suggesting immediate translatability to the clinic.