RESUMEN
Group 3 innate lymphoid cells (ILC3s) regulate immunity and inflammation, yet their role in cancer remains elusive. Here, we identify that colorectal cancer (CRC) manifests with altered ILC3s that are characterized by reduced frequencies, increased plasticity, and an imbalance with T cells. We evaluated the consequences of these changes in mice and determined that a dialog between ILC3s and T cells via major histocompatibility complex class II (MHCII) is necessary to support colonization with microbiota that subsequently induce type-1 immunity in the intestine and tumor microenvironment. As a result, mice lacking ILC3-specific MHCII develop invasive CRC and resistance to anti-PD-1 immunotherapy. Finally, humans with dysregulated intestinal ILC3s harbor microbiota that fail to induce type-1 immunity and immunotherapy responsiveness when transferred to mice. Collectively, these data define a protective role for ILC3s in cancer and indicate that their inherent disruption in CRC drives dysfunctional adaptive immunity, tumor progression, and immunotherapy resistance.
Asunto(s)
Neoplasias del Colon/inmunología , Neoplasias del Colon/terapia , Progresión de la Enfermedad , Inmunidad Innata , Inmunoterapia , Linfocitos/inmunología , Animales , Comunicación Celular/efectos de los fármacos , Plasticidad de la Célula/efectos de los fármacos , Neoplasias del Colon/microbiología , Heces/microbiología , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inmunidad Innata/efectos de los fármacos , Inflamación/inmunología , Inflamación/patología , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/microbiología , Enfermedades Inflamatorias del Intestino/patología , Intestinos/patología , Linfocitos/efectos de los fármacos , Ratones Endogámicos C57BL , Microbiota/efectos de los fármacos , Invasividad Neoplásica , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Donantes de TejidosRESUMEN
Carcinogenesis is a multistep process by which normal cells acquire genetic and epigenetic changes that result in cancer. In combination with host genetic susceptibility and environmental exposures, a prominent procarcinogenic role for the microbiota has recently emerged. In colorectal cancer (CRC), three nefarious microbes have been consistently linked to cancer development: (a) Colibactin-producing Escherichia coli initiates carcinogenic DNA damage, (b) enterotoxigenic Bacteroides fragilis promotes tumorigenesis via toxin-induced cell proliferation and tumor-promoting inflammation, and (c) Fusobacterium nucleatum enhances CRC progression through two adhesins, Fap2 and FadA, that promote proliferation and antitumor immune evasion and may contribute to metastases. Herein, we use these three prominent microbes to discuss the experimental evidence linking microbial activities to carcinogenesis and the specific mechanisms driving this stepwise process. Precisely defining mechanisms by which the microbiota impacts carcinogenesis at each stage is essential for developing microbiota-targeted strategies for the diagnosis, prognosis, and treatment of cancer.
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Carcinogénesis/genética , Transformación Celular Neoplásica/genética , Neoplasias Colorrectales/microbiología , Microbiota , Neoplasias Colorrectales/genética , Progresión de la Enfermedad , HumanosRESUMEN
Cancers arising in mucosal tissues account for a disproportionately large fraction of malignancies. Immunoglobulin G (IgG) and the neonatal Fc receptor for IgG (FcRn) have an important function in the mucosal immune system that we have now shown extends to the induction of CD8(+) T cell-mediated antitumor immunity. We demonstrate that FcRn within dendritic cells (DCs) was critical for homeostatic activation of mucosal CD8(+) T cells that drove protection against the development of colorectal cancers and lung metastases. FcRn-mediated tumor protection was driven by DCs activation of endogenous tumor-reactive CD8(+) T cells via the cross-presentation of IgG complexed antigens (IgG IC), as well as the induction of cytotoxicity-promoting cytokine secretion, particularly interleukin-12, both of which were independently triggered by the FcRn-IgG IC interaction in murine and human DCs. FcRn thus has a primary role within mucosal tissues in activating local immune responses that are critical for priming efficient anti-tumor immunosurveillance.
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Neoplasias Colorrectales/inmunología , Células Dendríticas/metabolismo , Regulación Neoplásica de la Expresión Génica , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunidad/genética , Receptores Fc/genética , Receptores Fc/metabolismo , Animales , Neoplasias Colorrectales/genética , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Citometría de Flujo , Humanos , Inmunidad Activa , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
In vitro data suggest that a subgroup of NLR proteins, including NLRP12, inhibits the transcription factor NF-κB, although physiologic and disease-relevant evidence is largely missing. Dysregulated NF-κB activity is associated with colonic inflammation and cancer, and we found Nlrp12(-/-) mice were highly susceptible to colitis and colitis-associated colon cancer. Polyps isolated from Nlrp12(-/-) mice showed elevated noncanonical NF-κB activation and increased expression of target genes that were associated with cancer, including Cxcl13 and Cxcl12. NLRP12 negatively regulated ERK and AKT signaling pathways in affected tumor tissues. Both hematopoietic- and nonhematopoietic-derived NLRP12 contributed to inflammation, but the latter dominantly contributed to tumorigenesis. The noncanonical NF-κB pathway was regulated upon degradation of TRAF3 and activation of NIK. NLRP12 interacted with both NIK and TRAF3, and Nlrp12(-/-) cells have constitutively elevated NIK, p100 processing to p52 and reduced TRAF3. Thus, NLRP12 is a checkpoint of noncanonical NF-κB, inflammation, and tumorigenesis.
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Transformación Celular Neoplásica/patología , Colitis/metabolismo , Colitis/patología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , FN-kappa B/metabolismo , Animales , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Quimiocina CXCL13/genética , Quimiocina CXCL13/metabolismo , Colitis/genética , Neoplasias del Colon/genética , Progresión de la Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Ratones , Ratones Endogámicos C57BL , FN-kappa B/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Factor 3 Asociado a Receptor de TNF/genética , Factor 3 Asociado a Receptor de TNF/metabolismo , Quinasa de Factor Nuclear kappa BRESUMEN
Carcinoembryonic antigen cell adhesion molecule like I (CEACAM1) is expressed on activated T cells and signals through either a long (L) cytoplasmic tail containing immune receptor tyrosine based inhibitory motifs, which provide inhibitory function, or a short (S) cytoplasmic tail with an unknown role. Previous studies on peripheral T cells show that CEACAM1-L isoforms predominate with little to no detectable CEACAM1-S isoforms in mouse and human. We show here that this was not the case in tissue resident T cells of intestines and gut associated lymphoid tissues, which demonstrated predominant expression of CEACAM1-S isoforms relative to CEACAM1-L isoforms in human and mouse. This tissue resident predominance of CEACAM1-S expression was determined by the intestinal environment where it served a stimulatory function leading to the regulation of T cell subsets associated with the generation of secretory IgA immunity, the regulation of mucosal commensalism, and defense of the barrier against enteropathogens.
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Antígeno Carcinoembrionario/inmunología , Inmunidad Mucosa/inmunología , Intestinos/inmunología , Linfocitos T/inmunología , Secuencias de Aminoácidos/genética , Secuencias de Aminoácidos/inmunología , Animales , Antígeno Carcinoembrionario/genética , Antígeno Carcinoembrionario/metabolismo , Citoplasma/genética , Citoplasma/inmunología , Citoplasma/metabolismo , Homeostasis , Inmunidad Mucosa/genética , Inmunoglobulina A/genética , Inmunoglobulina A/inmunología , Inmunoglobulina A/metabolismo , Mucosa Intestinal/metabolismo , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Activación de Linfocitos , Metagenoma/inmunología , Ratones , Ratones Endogámicos C57BL , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Isoformas de Proteínas , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/metabolismo , Linfocitos T/metabolismo , Tirosina/genética , Tirosina/inmunología , Tirosina/metabolismoRESUMEN
Fibrosis is a significant complication of intestinal disorders associated with microbial dysbiosis and pathobiont expansion, notably Crohn's disease (CD). Mechanisms that favor fibrosis are not well understood, and therapeutic strategies are limited. Here we demonstrate that colitis-susceptible Il10-deficient mice develop inflammation-associated fibrosis when monoassociated with adherent/invasive Escherichia coli (AIEC) that harbors the yersiniabactin (Ybt) pathogenicity island. Inactivation of Ybt siderophore production in AIEC nearly abrogated fibrosis development in inflamed mice. In contrast, inactivation of Ybt import through its cognate receptor FyuA enhanced fibrosis severity. This corresponded with increased colonic expression of profibrogenic genes prior to the development of histological disease, therefore suggesting causality. fyuA-deficient AIEC also exhibited greater localization within subepithelial tissues and fibrotic lesions that was dependent on Ybt biosynthesis and corresponded with increased fibroblast activation in vitro Together, these findings suggest that Ybt establishes a profibrotic environment in the host in the absence of binding to its cognate receptor and indicate a direct link between intestinal AIEC and the induction of inflammation-associated fibrosis.
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Colitis/microbiología , Escherichia coli/metabolismo , Fibrosis/etiología , Inflamación/microbiología , Interleucina-10/metabolismo , Fenoles/metabolismo , Tiazoles/metabolismo , Animales , Adhesión Bacteriana , Colitis/complicaciones , Colitis/patología , Regulación Bacteriana de la Expresión Génica , Vida Libre de Gérmenes , Humanos , Inflamación/patología , Interleucina-10/genética , Ratones , Ratones Noqueados , MutaciónRESUMEN
Probiotic and engineered microbe-based therapeutics are an emerging class of pharmaceutical agents. They represent a promising strategy for treating various chronic and inflammatory conditions by interacting with the host immune system and/or delivering therapeutic molecules. Here, we engineered a targeted probiotic yeast platform wherein Saccharomyces boulardii is designed to bind to abundant extracellular matrix proteins found within inflammatory lesions of the gastrointestinal tract through tunable antibody surface display. This approach enabled an additional 24-48 h of probiotic gut residence time compared to controls and 100-fold increased probiotic concentrations within the colon in preclinical models of ulcerative colitis in female mice. As a result, pharmacodynamic parameters including colon length, colonic cytokine expression profiles, and histological inflammation scores were robustly improved and restored back to healthy levels. Overall, these studies highlight the potential for targeted microbial therapeutics as a potential oral dosage form for the treatment of inflammatory bowel diseases.
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Colitis Ulcerosa , Colon , Modelos Animales de Enfermedad , Matriz Extracelular , Probióticos , Saccharomyces boulardii , Animales , Probióticos/administración & dosificación , Femenino , Ratones , Matriz Extracelular/metabolismo , Colitis Ulcerosa/terapia , Colitis Ulcerosa/microbiología , Colitis Ulcerosa/patología , Colon/microbiología , Colon/metabolismo , Colon/patología , Ratones Endogámicos C57BL , Colitis/terapia , Colitis/microbiología , Colitis/patología , Citocinas/metabolismo , HumanosRESUMEN
Escherichia coli isolates from inflammatory bowel disease (IBD) patients are often multidrug resistant, including to streptomycin. Streptomycin resistance (StrR) mutations can alter bacterial behavior, which may influence intestinal disease. We generated a spontaneous StrR strain of the intestinal adherent-invasive E. coli (AIEC) strain NC101. Whole-genome sequencing revealed a single missense mutation in rpsL that commonly confers StrR, rpsL-K43N. StrR NC101 exhibited a striking loss of aggregation and significantly increased motility, behaviors that can impact host-microbe interactions. Behavioral changes were associated with reduced transcription of csgA, encoding the biofilm component curli, and increased transcription of fliC, encoding flagellin. Scanning electron microscopy (SEM) detailed morphologic changes consistent with the observed alterations in multicellular behavior. Because intestinal E. coli isolates exhibit remarkable strain-specific differences, we generated spontaneous StrR mutants of 10 clinical E. coli phylotype B2 strains from patients with IBD, colorectal cancer, and urinary tract infection. Out of these 10 StrR clinical strains, two had altered colony morphology on Congo red agar (suggesting changes in extracellular products), and three had significant changes in motility. These changes were not associated with a particular rpsL mutation nor with the presence of virulence genes encoding the inflammation-associated E. coli metabolites yersiniabactin or colibactin. We conclude that common mutations in rpsL, which confer StrR, can differentially alter disease-associated phenotypes across intestinal E. coli strains. These findings highlight the heterogeneity among seemingly similar intestinal E. coli strains and reveal the need to carefully study the strain-specific effects of antibiotic resistance mutations, particularly when using these mutations during strain selection studies. IMPORTANCE We demonstrate that StrR, commonly acquired through a single point mutation in rpsL (a gene encoding part of the 30S bacterial ribosome), strikingly alters the morphology and behavior of a key intestinal AIEC strain, NC101. These changes include remarkably diminished aggregation and significantly increased motility, traits that are linked to AIEC-defining features and disease development. Phenotypic changes were heterogeneous among other StrR clinical E. coli strains, underscoring the need to evaluate the strain-specific effects of commonly acquired antibiotic resistance mutations. This is important, as the results of studies using mutant StrR Enterobacteriaceae strains (e.g., for cloning or in vivo selection) may be confounded beyond our demonstrated effects. Long term, these findings can help researchers better distinguish the contribution of specific E. coli traits to functional changes in the microbiota. Evaluating these strain-level differences could provide insight into the diversity of IBD symptoms and lead to improved therapies for microbiota-driven intestinal disorders.
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Infecciones por Escherichia coli , Enfermedades Inflamatorias del Intestino , Humanos , Estreptomicina/farmacología , Escherichia coli , Mutación , Mutación Puntual , Infecciones por Escherichia coli/microbiologíaRESUMEN
Background: Inflammatory bowel disease (IBD) patients experience recurrent episodes of intestinal inflammation and often follow an unpredictable disease course. Mucosal colonization with adherent-invasive Escherichia coli (AIEC) are believed to perpetuate intestinal inflammation. However, it remains unclear if the 24-year-old AIEC in-vitro definition fully predicts mucosal colonization in-vivo. To fill this gap, we have developed a novel molecular barcoding approach to distinguish strain variants in the gut and have integrated this approach to explore mucosal colonization of distinct patient-derived E. coli isolates in gnotobiotic mouse models of colitis. Results: Germ-free inflammation-susceptible interleukin-10-deficient (Il10-/-) and inflammation-resistant WT mice were colonized with a consortia of AIEC and non-AIEC strains, then given a murine fecal transplant to provide niche competition. E. coli strains isolated from human intestinal tissue were each marked with a unique molecular barcode that permits identification and quantification by barcode-targeted sequencing. 16S rRNA sequencing was used to evaluate the microbiome response to E. coli colonization. Our data reveal that specific AIEC and non-AIEC strains reproducibly colonize the intestinal mucosa of WT and Il10-/- mice. These E. coli expand in Il10-/- mice during inflammation and induce compositional dysbiosis to the microbiome in an inflammation-dependent manner. In turn, specific microbes co-evolve in inflamed mice, potentially diversifying E. coli colonization patterns. We observed no selectivity in E. coli colonization patterns in the fecal contents, indicating minimal selective pressure in this niche from host-microbe and interbacterial interactions. Because select AIEC and non-AIEC strains colonize the mucosa, this suggests the in vitro AIEC definition may not fully predict in vivo colonization potential. Further comparison of seven E. coli genomes pinpointed unique genomic features contained only in highly colonizing strains (two AIEC and two non-AIEC). Those colonization-associated features may convey metabolic advantages (e.g., iron acquisition and carbohydrate consumption) to promote efficient mucosal colonization. Conclusions: Our findings establish the in-vivo mucosal colonizer, not necessarily AIEC, as a principal dysbiosis driver through crosstalk with host and associated microbes. Furthermore, we highlight the utility of high-throughput screens to decode the in-vivo colonization dynamics of patient-derived bacteria in murine models.
RESUMEN
BACKGROUND: Inflammatory bowel disease (IBD) patients experience recurrent episodes of intestinal inflammation and often follow an unpredictable disease course. Mucosal colonization with adherent-invasive Escherichia coli (AIEC) are believed to perpetuate intestinal inflammation. However, it remains unclear if the 24-year-old AIEC in vitro definition fully predicts mucosal colonization in vivo. To fill this gap, we have developed a novel molecular barcoding approach to distinguish strain variants in the gut and have integrated this approach to explore mucosal colonization of distinct patient-derived E. coli isolates in gnotobiotic mouse models of colitis. RESULTS: Germ-free inflammation-susceptible interleukin-10-deficient (Il10-/-) and inflammation-resistant WT mice were colonized with a consortium of AIEC and non-AIEC strains, then given a murine fecal transplant to provide niche competition. E. coli strains isolated from human intestinal tissue were each marked with a unique molecular barcode that permits identification and quantification by barcode-targeted sequencing. 16S rRNA sequencing was used to evaluate the microbiome response to E. coli colonization. Our data reveal that specific AIEC and non-AIEC strains reproducibly colonize the intestinal mucosa of WT and Il10-/- mice. These E. coli expand in Il10-/- mice during inflammation and induce compositional dysbiosis to the microbiome in an inflammation-dependent manner. In turn, specific microbes co-evolve in inflamed mice, potentially diversifying E. coli colonization patterns. We observed no selectivity in E. coli colonization patterns in the fecal contents, indicating minimal selective pressure in this niche from host-microbe and interbacterial interactions. Because select AIEC and non-AIEC strains colonize the mucosa, this suggests the in vitro AIEC definition may not fully predict in vivo colonization potential. Further comparison of seven E. coli genomes pinpointed unique genomic features contained only in highly colonizing strains (two AIEC and two non-AIEC). Those colonization-associated features may convey metabolic advantages (e.g., iron acquisition and carbohydrate consumption) to promote efficient mucosal colonization. CONCLUSIONS: Our findings establish the in vivo mucosal colonizer, not necessarily AIEC, as a principal dysbiosis driver through crosstalk with host and associated microbes. Furthermore, we highlight the utility of high-throughput screens to decode the in vivo colonization dynamics of patient-derived bacteria in murine models. Video Abstract.
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Infecciones por Escherichia coli , Microbioma Gastrointestinal , Animales , Humanos , Ratones , Adulto Joven , Disbiosis/complicaciones , Escherichia coli/genética , Infecciones por Escherichia coli/microbiología , Inflamación/metabolismo , Interleucina-10 , Mucosa Intestinal/microbiología , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismoRESUMEN
Increasing evidence implicates the tumor microbiota as a factor that can influence cancer progression. In patients with colorectal cancer (CRC), we found that pre-resection antibiotics targeting anaerobic bacteria substantially improved disease-free survival by 25.5%. For mouse studies, we designed an antibiotic silver-tinidazole complex encapsulated in liposomes (LipoAgTNZ) to eliminate tumor-associated bacteria in the primary tumor and liver metastases without causing gut microbiome dysbiosis. Mouse CRC models colonized by tumor-promoting bacteria (Fusobacterium nucleatum spp.) or probiotics (Escherichia coli Nissle spp.) responded to LipoAgTNZ therapy, which enabled more than 70% long-term survival in two F. nucleatum-infected CRC models. The antibiotic treatment generated microbial neoantigens that elicited anti-tumor CD8+ T cells. Heterologous and homologous bacterial epitopes contributed to the immunogenicity, priming T cells to recognize both infected and uninfected tumors. Our strategy targets tumor-associated bacteria to elicit anti-tumoral immunity, paving the way for microbiome-immunotherapy interventions.
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Nucleotide-binding domain leucine-rich repeat (NLR) proteins are regulators of inflammation and immunity. Although first described 8 y ago, a physiologic role for NLRP12 has remained elusive until now. We find that murine Nlrp12, an NLR linked to atopic dermatitis and hereditary periodic fever in humans, is prominently expressed in dendritic cells (DCs) and neutrophils. Nlrp12-deficient mice exhibit attenuated inflammatory responses in two models of contact hypersensitivity that exhibit features of allergic dermatitis. This cannot be attributed to defective Ag processing/presentation, inflammasome activation, or measurable changes in other inflammatory cytokines. Rather, Nlrp12(-/-) DCs display a significantly reduced capacity to migrate to draining lymph nodes. Both DCs and neutrophils fail to respond to chemokines in vitro. These findings indicate that NLRP12 is important in maintaining neutrophils and peripheral DCs in a migration-competent state.
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Quimiotaxis de Leucocito/inmunología , Células Dendríticas/inmunología , Dermatitis por Contacto/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Células Mieloides/inmunología , Animales , Células Dendríticas/metabolismo , Dermatitis por Contacto/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Mieloides/metabolismo , Reacción en Cadena de la PolimerasaRESUMEN
Inflammatory bowel disease (IBD) is a significant global health problem that involves chronic intestinal inflammation and can involve severe comorbidities, including intestinal fibrosis and inflammation-associated colorectal cancer (CRC). Disease-associated alterations to the intestinal microbiota often include fecal enrichment of Enterobacteriaceae, which are strongly implicated in IBD development. This dysbiosis of intestinal flora accompanies changes in microbial metabolites, shaping host:microbe interactions and disease risk. While there have been numerous studies linking specific bacterial taxa with IBD development, our understanding of microbial function in the context of IBD is limited. Several classes of microbial metabolites have been directly implicated in IBD disease progression, including bacterial siderophores and genotoxins. Yet, our microbiota still harbors thousands of uncharacterized microbial products. In-depth discovery and characterization of disease-associated microbial metabolites is necessary to target these products in IBD treatment strategies. Towards improving our understanding of microbiota metabolites in IBD, it is important to recognize how host relevant factors influence microbiota function. For example, changes in host inflammation status, metal availability, interbacterial community structure, and xenobiotics all play an important role in shaping gut microbial ecology. In this minireview, we outline how each of these factors influences gut microbial function, with a specific focus on IBD-associated Enterobacteriaceae metabolites. Importantly, we discuss how altering the intestinal microenvironment could improve the treatment of intestinal inflammation and associated disorders, like intestinal fibrosis and CRC.
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Microbioma Gastrointestinal , Enfermedades Inflamatorias del Intestino , Bacterias , Disbiosis/microbiología , Enterobacteriaceae , Fibrosis , Humanos , Inflamación/microbiología , Enfermedades Inflamatorias del Intestino/microbiologíaRESUMEN
Introduction: In colitis, macrophage functionality is altered compared to normal homeostatic conditions. Loss of IL-10 signaling results in an inappropriate chronic inflammatory response to bacterial stimulation. It remains unknown if inhibition of bromodomain and extra-terminal domain (BET) proteins alters usage of DNA regulatory elements responsible for driving inflammatory gene expression. We determined if the BET inhibitor, (+)-JQ1, could suppress inflammatory activation of macrophages in Il10-/- mice. Methods: We performed ATAC-seq and RNA-seq on Il10-/- bone marrow-derived macrophages (BMDMs) cultured in the presence and absence of lipopolysaccharide (LPS) with and without treatment with (+)-JQ1 and evaluated changes in chromatin accessibility and gene expression. Germ-free Il10-/- mice were treated with (+)-JQ1, colonized with fecal slurries and underwent histological and molecular evaluation 14-days post colonization. Results: Treatment with (+)-JQ1 suppressed LPS-induced changes in chromatin at distal regulatory elements associated with inflammatory genes, particularly in regions that contain motifs for AP-1 and IRF transcription factors. This resulted in attenuation of inflammatory gene expression. Treatment with (+)-JQ1 in vivo resulted in a mild reduction in colitis severity as compared with vehicle-treated mice. Conclusion: We identified the mechanism of action associated with a new class of compounds that may mitigate aberrant macrophage responses to bacteria in colitis.
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Colitis , Microbiota , Animales , Cromatina/genética , Cromatina/metabolismo , Colitis/inducido químicamente , Colitis/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Ratones , Proteínas del Tejido Nervioso , Receptores de Superficie Celular , Factores de Transcripción/metabolismoRESUMEN
Evidence supports involvement of microflora in the transition of chronic inflammation to neoplasia. We investigated the protective efficacy of the probiotic VSL#3 in a model of colitis-associated colorectal cancer. Chronic colitis was induced in Sprague-Dawley rats by administration of trinitrobenzene sulfonic acid (TNBS), followed 6 wk later by systemic reactivation. To induce colitis-associated dysplasia and cancer, the animals received TNBS (intravenously) twice a week for 10 wk. One group received VSL#3 in drinking water from 1 wk before colitis induction until death. The colons were examined for damage and presence of dysplasia or cancer. Samples were analyzed for cell proliferation and apoptosis, vitamin D receptor (VDR) expression, angiogenic factors, and presence of alkaline sphingomyelinase or phosphatase. Microbial community composition was evaluated by terminal restriction fragment-length polymorphism analysis of the bacterial 16S rRNA gene. None of the probiotic-treated animals developed carcinoma, and no high-grade dysplasia was found in either the proximal or mid colon. In contrast, 29% of the animals in the control group developed carcinoma in one or more regions of the colon. VSL#3-treated animals had significantly less damage than the vehicle treated-controls in all areas of the colon, and this correlated with decreased richness and diversity of the mucosally adherent microbiota. Treatment with the probiotic increased the antiangiogenic factor angiostatin, VDR expression, and alkaline sphingomyelinase. We concluded that pretreatment with the probiotic VSL#3 can attenuate various inflammatory-associated parameters, delaying transition to dysplasia and cancer, thus offering its potential therapeutic use in patients with long-standing colitis.
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Colitis/terapia , Neoplasias Colorrectales/prevención & control , Lesiones Precancerosas/terapia , Probióticos/farmacología , Fosfatasa Alcalina/metabolismo , Angiostatinas/metabolismo , Animales , Bifidobacterium , Enfermedad Crónica , Colitis/inmunología , Colitis/microbiología , Colon/metabolismo , Colon/microbiología , Colon/patología , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/microbiología , Modelos Animales de Enfermedad , Endostatinas/metabolismo , Lactobacillus , Masculino , Metagenoma , Lesiones Precancerosas/inmunología , Lesiones Precancerosas/microbiología , Ratas , Ratas Sprague-Dawley , Receptores de Calcitriol/metabolismo , Streptococcus , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Inflammatory bowel diseases (IBDs) and inflammation-associated colorectal cancer (CRC) are linked to blooms of adherent-invasive Escherichia coli (AIEC) in the intestinal microbiota. AIEC are functionally defined by their ability to adhere/invade epithelial cells and survive/replicate within macrophages. Changes in micronutrient availability can alter AIEC physiology and interactions with host cells. Thus, culturing AIEC for mechanistic investigations often involves precise nutrient formulation. We observed that the pro-inflammatory and pro-carcinogenic AIEC strain NC101 failed to grow in minimal media (MM). We hypothesized that NC101 was unable to synthesize a vital micronutrient normally found in the host gut. Through nutrient supplementation studies, we identified that NC101 is a nicotinic acid (NA) auxotroph. NA auxotrophy was not observed in the other non-toxigenic E. coli or AIEC strains we tested. Sequencing revealed NC101 has a missense mutation in nadA, a gene encoding quinolinate synthase A that is important for de novo nicotinamide adenine dinucleotide (NAD) biosynthesis. Correcting the identified nadA point mutation restored NC101 prototrophy without impacting AIEC function, including motility and AIEC-defining survival in macrophages. Our findings, along with the generation of a prototrophic NC101 strain, will greatly enhance the ability to perform in vitro functional studies that are needed for mechanistic investigations on the role of intestinal E. coli in digestive disease.
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The complex reservoir of metabolite-producing bacteria in the gastrointestinal tract contributes tremendously to human health and disease. Bacterial composition, and by extension gut metabolomic composition, is undoubtably influenced by the use of modern antibiotics. Herein, we demonstrate that polymyxin B, a last resort antibiotic, influences the production of the genotoxic metabolite colibactin from adherent-invasive Escherichia coli (AIEC) NC101. Colibactin can promote colorectal cancer through DNA double stranded breaks and interstrand cross-links. While the structure and biosynthesis of colibactin have been elucidated, chemical-induced regulation of its biosynthetic gene cluster and subsequent production of the genotoxin by E. coli are largely unexplored. Using a multiomic approach, we identified that polymyxin B stress enhances the abundance of colibactin biosynthesis proteins (Clb's) in multiple pks+ E. coli strains, including pro-carcinogenic AIEC, NC101; the probiotic strain, Nissle 1917; and the antibiotic testing strain, ATCC 25922. Expression analysis via qPCR revealed that increased transcription of clb genes likely contributes to elevated Clb protein levels in NC101. Enhanced production of Clb's by NC101 under polymyxin stress matched an increased production of the colibactin prodrug motif, a proxy for the mature genotoxic metabolite. Furthermore, E. coli with a heightened tolerance for polymyxin induced greater mammalian DNA damage, assessed by quantification of γH2AX staining in cultured intestinal epithelial cells. This study establishes a key link between the polymyxin B stress response and colibactin production in pks+ E. coli. Ultimately, our findings will inform future studies investigating colibactin regulation and the ability of seemingly innocuous commensal microbes to induce host disease.
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Farmacorresistencia Bacteriana/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Péptidos/efectos de los fármacos , Polimixinas/farmacología , Animales , Evolución Biológica , Línea Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Genes Bacterianos/efectos de los fármacos , Familia de Multigenes/efectos de los fármacos , Mutágenos/metabolismo , Péptido Sintasas/genética , Péptidos/metabolismo , Sintasas Poliquetidas/genética , Policétidos/metabolismo , Ratas , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Adherent-invasive Escherichia coli (AIEC) is a pathovar linked to inflammatory bowel diseases (IBD), especially Crohn's disease, and colorectal cancer. AIEC are genetically diverse, and in the absence of a universal molecular signature, are defined by in vitro functional attributes. The relative ability of difference AIEC strains to colonize, persist, and induce inflammation in an IBD-susceptible host is unresolved. To evaluate strain-level variation among tissue-associated E. coli in the intestines, we develop a long-read sequencing approach to identify AIEC by strain that excludes host DNA. We use this approach to distinguish genetically similar strains and assess their fitness in colonizing the intestine. Here we have assembled complete genomes using long-read nanopore sequencing for a model AIEC strain, NC101, and seven strains isolated from the intestinal mucosa of Crohn's disease and non-Crohn's tissues. We show these strains can colonize the intestine of IBD susceptible mice and induce inflammatory cytokines from cultured macrophages. We demonstrate that these strains can be quantified and distinguished in the presence of 99.5% mammalian DNA and from within a fecal population. Analysis of global genomic structure and specific sequence variation within the ribosomal RNA operon provides a framework for efficiently tracking strain-level variation of closely-related E. coli and likely other commensal/pathogenic bacteria impacting intestinal inflammation in experimental settings and IBD patients.
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Enfermedad de Crohn/microbiología , Escherichia coli/genética , Mucosa Intestinal/microbiología , Animales , Adhesión Bacteriana/genética , Enfermedad de Crohn/patología , Escherichia coli/aislamiento & purificación , Humanos , Inflamación/microbiología , Inflamación/patología , Mucosa Intestinal/patología , RatonesRESUMEN
Iron deficiency, a common comorbidity of gastrointestinal inflammatory disorders such as inflammatory bowel diseases (IBD), is often treated with oral iron supplementation. However, the safety of oral iron supplementation remains controversial because of its association with exacerbated disease activity in a subset of IBD patients. Because iron modulates bacterial growth and function, one possible mechanism by which iron may exacerbate inflammation in susceptible hosts is by modulating the intestinal microbiota. We, therefore, investigated the impact of dietary iron on the intestinal microbiota, utilizing the conventionalization of germ-free mice as a model of a microbial community in compositional flux to recapitulate the instability of the IBD-associated intestinal microbiota. Our findings demonstrate that altering intestinal iron availability during community assembly modulated the microbiota in non-inflamed wild type (WT) and colitis-susceptible interleukin-10-deficient (Il10-/-) mice. Depletion of luminal iron availability promoted luminal compositional changes associated with dysbiotic states irrespective of host genotype, including an expansion of Enterobacteriaceae such as Escherichia coli. Mechanistic in vitro growth competitions confirmed that high-affinity iron acquisition systems in E. coli enhance its abundance over other bacteria in iron-restricted conditions, thereby enabling pathobiont iron scavenging during dietary iron restriction. In contrast, distinct luminal community assembly was observed with dietary iron supplementation in WT versus Il10-/- mice, suggesting that the effects of increased iron on the microbiota differ with host inflammation status. Taken together, shifts in dietary iron intake during community assembly modulate the ecological structure of the intestinal microbiota and is dependent on host genotype and inflammation status.