RESUMO
Paneth cells are the primary source of C-type lysozyme, a ß-1,4-N-acetylmuramoylhydrolase that enzymatically processes bacterial cell walls. Paneth cells are normally present in human cecum and ascending colon, but are rarely found in descending colon and rectum; Paneth cell metaplasia in this region and aberrant lysozyme production are hallmarks of inflammatory bowel disease (IBD) pathology. Here, we examined the impact of aberrant lysozyme production in colonic inflammation. Targeted disruption of Paneth cell lysozyme (Lyz1) protected mice from experimental colitis. Lyz1-deficiency diminished intestinal immune responses to bacterial molecular patterns and resulted in the expansion of lysozyme-sensitive mucolytic bacteria, including Ruminococcus gnavus, a Crohn's disease-associated pathobiont. Ectopic lysozyme production in colonic epithelium suppressed lysozyme-sensitive bacteria and exacerbated colitis. Transfer of R. gnavus into Lyz1-/- hosts elicited a type 2 immune response, causing epithelial reprograming and enhanced anti-colitogenic capacity. In contrast, in lysozyme-intact hosts, processed R. gnavus drove pro-inflammatory responses. Thus, Paneth cell lysozyme balances intestinal anti- and pro-inflammatory responses, with implications for IBD.
Assuntos
Clostridiales/imunologia , Colite Ulcerativa/patologia , Muramidase/genética , Muramidase/metabolismo , Celulas de Paneth/metabolismo , Animais , Clostridiales/genética , Colite Ulcerativa/microbiologia , Doença de Crohn/patologia , Feminino , Microbioma Gastrointestinal/genética , Células Caliciformes/citologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Transcrição STAT6/genéticaRESUMO
Paneth cells (PCs), a specialized secretory cell type in the small intestine, are increasingly recognized as having an essential role in host responses to microbiome and environmental stresses. Whether and how commensal and pathogenic microbes modify PC composition to modulate inflammation remain unclear. Using newly developed PC-reporter mice under conventional and gnotobiotic conditions, we determined PC transcriptomic heterogeneity in response to commensal and invasive microbes at single cell level. Infection expands the pool of CD74+ PCs, whose number correlates with auto or allogeneic inflammatory disease progressions in mice. Similar correlation was found in human inflammatory disease tissues. Infection-stimulated cytokines increase production of reactive oxygen species (ROS) and expression of a PC-specific mucosal pentraxin (Mptx2) in activated PCs. A PC-specific ablation of MyD88 reduced CD74+ PC population, thus ameliorating pathogen-induced systemic disease. A similar phenotype was also observed in mice lacking Mptx2. Thus, infection stimulates expansion of a PC subset that influences disease progression.
Assuntos
Microbiota , Celulas de Paneth , Humanos , Animais , Camundongos , Celulas de Paneth/metabolismo , Celulas de Paneth/patologia , Intestino Delgado , Inflamação/patologia , Citocinas/metabolismoRESUMO
Lysozyme is a ß-1,4-glycosidase that hydrolyzes the polysaccharide backbone of bacterial cell walls. With an additional bactericidal function mediated by a separate protein domain, lysozyme is considered a uniquely important antimicrobial molecule contributing to the host's innate immune response to infection. Elevated lysozyme production is found in various inflammatory conditions while patients with genetic risks for inflammatory bowel diseases demonstrate abnormal lysozyme expression, granule packaging, and secretion in Paneth cells. However, it remains unclear how a gain- or loss-of-function in host lysozyme may impact the host inflammatory responses to pathogenic infection. We challenged Lyz1-/- and ectopic Lyz1-expressing (Villin-Lyz1TG) mice with S. Typhimurium and then comprehensively assessed the inflammatory disease progression. We conducted proteomics analysis to identify molecules derived from human lysozyme-mediated processing of live Salmonella. We examined the barrier-impairing effects of these identified molecules in human intestinal epithelial cell monolayer and enteroids. Lyz1-/- mice are protected from infection in terms of morbidity, mortality, and barrier integrity, whereas Villin-Lyz1TG mice demonstrate exacerbated infection and inflammation. The growth and invasion of Salmonella in vitro are not affected by human or chicken lysozyme, whereas lysozyme encountering of live Salmonella stimulates the release of barrier-disrupting factors, InvE-sipC and Lpp1, which directly or indirectly impair the tight junctions. The direct engagement of host intestinal lysozyme with an enteric pathogen such as Salmonella promotes the release of virulence factors that are barrier-impairing and pro-inflammatory. Controlling lysozyme function may help alleviate the inflammatory progression.
Assuntos
Muramidase , Salmonella typhimurium , Muramidase/metabolismo , Animais , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Camundongos , Humanos , Infecções por Salmonella/microbiologia , Infecções por Salmonella/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Camundongos Knockout , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas dos MicrofilamentosRESUMO
BACKGROUND: Ruminococcus gnavus is a mucolytic commensal bacterium whose increased gut colonization has been associated with chronic inflammatory and metabolic diseases in humans. Whether R. gnavus metabolites can modulate host intestinal physiology remains largely understudied. METHODS: We performed untargeted metabolomic and bulk RNA sequencing analyses using R. gnavus mono-colonization in germ free mice. Based on transcriptome-metabolome correlations, we tested the impact of specific arginine metabolites on intestinal epithelial production of nitric oxide (NO) and examined the effect of NO on the growth of various strains of R. gnavus in vitro and in Nos2-deficient mice. RESULTS: R. gnavus produces specific arginine, tryptophan and tyrosine metabolites, some of which are regulated by the environmental richness of sialic acid and mucin. R. gnavus colonization promotes expression of amino acid transporters and enzymes involved in metabolic flux of arginine and associated metabolites into NO. R. gnavus induced elevated levels of Nitric Oxide Synthase 2 (NOS2) while Nos2 ablation resulted in R. gnavus expansion in vivo. The growth of various R. gnavus strains can be inhibited by NO. Specific R. gnavus metabolites modulate intestinal epithelial cell NOS2 abundance and reduce epithelial barrier function at higher concentrations. CONCLUSIONS: Intestinal colonization and interaction with R. gnavus are partially regulated by an arginine-NO metabolic pathway, whereby a balanced control by the gut epithelium may restrain R. gnavus growth in healthy individuals. Disruption in this arginine metabolic regulation will contribute to the expansion and blooming of R. gnavus.
RESUMO
Although Wnt signaling is clearly important for the intestinal epithelial homeostasis, the relevance of various sources of Wnt ligands themselves remains incompletely understood. Blocking the release of Wnt in distinct stromal cell types suggests obligatory functions of several stromal cell sources and yields different observations. The physiological contribution of epithelial Wnt to tissue homeostasis remains unclear. We show here that blocking epithelial Wnts affects colonic Reg4+ epithelial cell differentiation and impairs colonic epithelial regeneration after injury in mice. Single-cell RNA analysis of intestinal stroma showed that the majority of Wnt-producing cells were contained in transgelin (Tagln+) and smooth muscle actin α2 (Acta2+) expressing populations. We genetically attenuated Wnt production from these stromal cells using Tagln-Cre and Acta2-CreER drivers, and found that blockage of Wnt release from either epithelium or Tagln+ and Acta2+ stromal cells impaired colonic epithelial healing after chemical-induced injury. Aggregated blockage of Wnt release from both epithelium and Tagln+ or Acta2+ stromal cells drastically diminished epithelial repair, increasing morbidity and mortality. These results from two uncharacterized stromal populations suggested that colonic recovery from colitis-like injury depends on multiple Wnt-producing sources.
Assuntos
Actinas/metabolismo , Colite Ulcerativa/metabolismo , Mucosa Intestinal/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Proteína Wnt3A/metabolismo , Cicatrização , Actinas/genética , Animais , Células Cultivadas , Colo/citologia , Colo/metabolismo , Colo/fisiologia , Mucosa Intestinal/citologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/genética , Proteínas Musculares/genética , Proteínas Associadas a Pancreatite/genética , Proteínas Associadas a Pancreatite/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/metabolismo , Proteína Wnt3A/genéticaRESUMO
Within the intestinal epithelium, regulation of intracellular protein and vesicular trafficking is of utmost importance for barrier maintenance, immune responses, and tissue polarity. RAB11A is a small GTPase that mediates the anterograde transport of protein cargos to the plasma membrane. Loss of RAB11A-dependent trafficking in mature intestinal epithelial cells results in increased epithelial proliferation and nuclear accumulation of Yes-associated protein (YAP), a key Hippo-signaling transducer that senses cell-cell contacts and regulates tissue growth. However, it is unclear how RAB11A regulates YAP intracellular localizations. In this report, we examined the relationship of RAB11A to epithelial junctional complexes, YAP, and the associated consequences on colonic epithelial tissue repair. We found that RAB11A controls the biochemical associations of YAP with multiple components of adherens and tight junctions, including α-catenin, ß-catenin, and Merlin, a tumor suppressor. In the absence of RAB11A and Merlin, we observed enhanced YAP-ß-catenin complex formation and nuclear translocation. Upon chemical injury to the intestine, mice deficient in RAB11A were found to have reduced epithelial integrity, decreased YAP localization to adherens and tight junctions, and increased nuclear YAP accumulation in the colon epithelium. Thus, RAB11A-regulated trafficking regulates the Hippo-YAP signaling pathway for rapid reparative response after tissue injury.
Assuntos
Proteínas de Ciclo Celular/genética , Colite/genética , Neurofibromina 2/genética , Fatores de Transcrição/genética , beta Catenina/genética , Proteínas rab de Ligação ao GTP/genética , Junções Aderentes/genética , Animais , Células CACO-2 , Proliferação de Células/genética , Colite/induzido quimicamente , Colite/patologia , Colo/crescimento & desenvolvimento , Colo/patologia , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Epitélio/crescimento & desenvolvimento , Epitélio/patologia , Humanos , Camundongos , Junções Íntimas/genética , alfa Catenina/genéticaRESUMO
A number of studies have examined the effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3 ) on intestinal inflammation driven by immune cells, while little information is currently available about its impact on inflammation caused by intestinal epithelial cell (IEC) defects. Mice lacking IEC-specific Rab11a a recycling endosome small GTPase resulted in increased epithelial cell production of inflammatory cytokines, notably IL-6 and early onset of enteritis. To determine whether vitamin D supplementation may benefit hosts with epithelial cell-originated mucosal inflammation, we evaluated in vivo effects of injected 1,25(OH)2 D3 or dietary supplement of a high dose of vitamin D on the gut phenotypes of IEC-specific Rab11a knockout mice (Rab11aΔIEC ). 1,25(OH)2 D3 administered at 25 ng, two doses per mouse, by intraperitoneal injection, reduced inflammatory cytokine production in knockout mice compared to vehicle-injected mice. Remarkably, feeding mice with dietary vitamin D supplementation at 20,000 IU/kg spanning fetal and postnatal developmental stages led to improved bodyweights, reduced immune cell infiltration, and decreased inflammatory cytokines. We found that these vitamin D effects were accompanied by decreased NF-κB (p65) in the knockout intestinal epithelia, reduced tissue-resident macrophages, and partial restoration of epithelial morphology. Our study suggests that dietary vitamin D supplementation may prevent and limit intestinal inflammation in hosts with high susceptibility to chronic inflammation.
Assuntos
Células Epiteliais/efeitos dos fármacos , Inflamação/tratamento farmacológico , Intestinos/efeitos dos fármacos , Vitamina D/análogos & derivados , Vitamina D/farmacologia , Animais , Citocinas/metabolismo , Dieta , Suplementos Nutricionais , Mucosa Intestinal/efeitos dos fármacos , CamundongosRESUMO
BACKGROUND: Lactobacillus rhamnosus GG (LGG) is the most widely used probiotic, but the mechanisms underlying its beneficial effects remain unresolved. Previous studies typically inoculated LGG in hosts with established gut microbiota, limiting the understanding of specific impacts of LGG on host due to numerous interactions among LGG, commensal microbes, and the host. There has been a scarcity of studies that used gnotobiotic animals to elucidate LGG-host interaction, in particular for gaining specific insights about how it modifies the metabolome. To evaluate whether LGG affects the metabolite output of pathobionts, we inoculated with LGG gnotobiotic mice containing Propionibacterium acnes, Turicibacter sanguinis, and Staphylococcus aureus (PTS). RESULTS: 16S rRNA sequencing of fecal samples by Ion Torrent and MinION platforms showed colonization of germ-free mice by PTS or by PTS plus LGG (LTS). Although the body weights and feeding rates of mice remained similar between PTS and LTS groups, co-associating LGG with PTS led to a pronounced reduction in abundance of P. acnes in the gut. Addition of LGG or its secretome inhibited P. acnes growth in culture. After optimizing procedures for fecal metabolite extraction and metabolomic liquid chromatography-mass spectrometry analysis, unsupervised and supervised multivariate analyses revealed a distinct separation among fecal metabolites of PTS, LTS, and germ-free groups. Variables-important-in-projection scores showed that LGG colonization robustly diminished guanine, ornitihine, and sorbitol while significantly elevating acetylated amino acids, ribitol, indolelactic acid, and histamine. In addition, carnitine, betaine, and glutamate increased while thymidine, quinic acid and biotin were reduced in both PTS and LTS groups. Furthermore, LGG association reduced intestinal mucosal expression levels of inflammatory cytokines, such as IL-1α, IL-1ß and TNF-α. CONCLUSIONS: LGG co-association had a negative impact on colonization of P. acnes, and markedly altered the metabolic output and inflammatory response elicited by pathobionts.
Assuntos
Infecções por Bactérias Gram-Positivas/microbiologia , Lacticaseibacillus rhamnosus/metabolismo , Probióticos/administração & dosagem , Animais , Citocinas/genética , Citocinas/metabolismo , Feminino , Firmicutes/crescimento & desenvolvimento , Firmicutes/fisiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Vida Livre de Germes , Infecções por Bactérias Gram-Positivas/genética , Infecções por Bactérias Gram-Positivas/metabolismo , Humanos , Lacticaseibacillus rhamnosus/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Propionibacterium acnes/crescimento & desenvolvimento , Propionibacterium acnes/fisiologia , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/fisiologiaRESUMO
NOD2 was the first susceptibility gene identified for Crohn's disease (CD), one of the major forms of inflammatory bowel disease (IBD). The field of NOD2 research has opened up many questions critical to understanding the complexities of microbiota-host interactions. In addition to sensing its specific bacterial components as a cytosolic pattern recognition receptor, NOD2 also appears to shape the colonization of intestinal microbiota. Activated NOD2 triggers downstream signaling cascades exampled by the NF-κB pathway to induce antimicrobial activities, however, defective or loss of NOD2 functions incur a similarly activated inflammatory response. Additional studies have identified the involvement of NOD2 in protection against non-microbiota-related intestinal damages as well as extraintestinal infections. We survey recent molecular and genetic studies of NOD2-mediated bacterial sensing and immunological modulation, and integrate evidence to suggest a highly reciprocal but still poorly understood cross talk between enteric microbiota and host cells.
Assuntos
Doença de Crohn/metabolismo , Regulação da Expressão Gênica/fisiologia , Mucosa Intestinal/metabolismo , Microbiota/fisiologia , Proteína Adaptadora de Sinalização NOD2/metabolismo , Doença de Crohn/patologia , Progressão da Doença , Homeostase , Humanos , Intestinos/microbiologia , Intestinos/patologia , Proteína Adaptadora de Sinalização NOD2/genéticaRESUMO
BACKGROUND & AIMS: Lacticaseibacillus rhamnosus GG (LGG) is the world's most consumed probiotic but its mechanism of action on intestinal permeability and differentiation along with its interactions with an essential source of signaling metabolites, dietary tryptophan (trp), are unclear. METHODS: Untargeted metabolomic and transcriptomic analyses were performed in LGG monocolonized germ-free mice fed trp-free or -sufficient diets. LGG-derived metabolites were profiled in vitro under anaerobic and aerobic conditions. Multiomic correlations using a newly developed algorithm discovered novel metabolites tightly linked to tight junction and cell differentiation genes whose abundances were regulated by LGG and dietary trp. Barrier-modulation by these metabolites were functionally tested in Caco2 cells, mouse enteroids, and dextran sulfate sodium experimental colitis. The contribution of these metabolites to barrier protection is delineated at specific tight junction proteins and enterocyte-promoting factors with gain and loss of function approaches. RESULTS: LGG, strictly with dietary trp, promotes the enterocyte program and expression of tight junction genes, particularly Ocln. Functional evaluations of fecal and serum metabolites synergistically stimulated by LGG and trp revealed a novel vitamin B3 metabolism pathway, with methylnicotinamide (MNA) unexpectedly being the most robust barrier-protective metabolite in vitro and in vivo. Reduced serum MNA is significantly associated with increased disease activity in patients with inflammatory bowel disease. Exogenous MNA enhances gut barrier in homeostasis and robustly promotes colonic healing in dextran sulfate sodium colitis. MNA is sufficient to promote intestinal epithelial Ocln and RNF43, a master inhibitor of Wnt. Blocking trp or vitamin B3 absorption abolishes barrier recovery in vivo. CONCLUSIONS: Our study uncovers a novel LGG-regulated dietary trp-dependent production of MNA that protects the gut barrier against colitis.
Assuntos
Colite , Lacticaseibacillus rhamnosus , Probióticos , Triptofano , Animais , Lacticaseibacillus rhamnosus/metabolismo , Lacticaseibacillus rhamnosus/fisiologia , Triptofano/metabolismo , Camundongos , Humanos , Células CACO-2 , Probióticos/administração & dosagem , Colite/metabolismo , Colite/patologia , Mucosa Intestinal/metabolismo , Enterócitos/metabolismo , Sulfato de Dextrana , Niacinamida/farmacologia , Niacinamida/metabolismo , Junções Íntimas/metabolismo , Masculino , Modelos Animais de Doenças , Proteínas de Junções Íntimas/metabolismoRESUMO
As a highly regenerative organ, the intestine is a promising source for cellular reprogramming for replacing lost pancreatic ß cells in diabetes. Gut enterochromaffin cells can be converted to insulin-producing cells by forkhead box O1 (FoxO1) ablation, but their numbers are limited. In this study, we report that insulin-immunoreactive cells with Paneth/goblet cell features are present in human fetal intestine. Accordingly, lineage-tracing experiments show that, upon genetic or pharmacologic FoxO1 ablation, the Paneth/goblet lineage can also undergo conversion to the insulin lineage. We designed a screening platform in gut organoids to accurately quantitate ß-like cell reprogramming and fine-tune a combination treatment to increase the efficiency of the conversion process in mice and human adult intestinal organoids. We identified a triple blockade of FOXO1, Notch, and TGF-ß that, when tested in insulin-deficient streptozotocin (STZ) or NOD diabetic animals, resulted in near normalization of glucose levels, associated with the generation of intestinal insulin-producing cells. The findings illustrate a therapeutic approach for replacing insulin treatment in diabetes.
Assuntos
Diabetes Mellitus , Células Secretoras de Insulina , Humanos , Camundongos , Animais , Proteína Forkhead Box O1/genética , Fatores de Transcrição Forkhead/genética , Camundongos Endogâmicos NOD , Insulina/genéticaRESUMO
Paneth cells are residents of the intestinal epithelium. Abnormal appearance of Paneth cells has been widely documented in non-intestinal tissues within the digestive tract and even observed in non-gastrointestinal organs. Although metaplastic Paneth cells are part of the overarching pathology of intestinal metaplasia (IM), only a fraction of intestinal metaplastic lesions contain Paneth cells. We survey literature documenting metaplastic Paneth cells to gain insights into mechanism underlying their etiologic development as well as their potential relevance to human health. A synthesized view from this study suggests that the emergence of metaplastic Paneth cells at extra-intestinal mucosal sites likely represents a protective, anti-bacterial, and inflammatory response evoked by an altered microbial activity.
RESUMO
The effects of polarized membrane trafficking in mature epithelial tissue on cell growth and cancer progression have not been fully explored in vivo. A majority of colorectal cancers have reduced and mislocalized Rab11, a small GTPase dedicated to trafficking of recycling endosomes. Patients with low Rab11 protein expression have poor survival rates. Using genetic models across species, we show that intact recycling endosome function restrains aberrant epithelial growth elicited by APC or RAS mutations. Loss of Rab11 protein led to epithelial dysplasia in early animal development and synergized with oncogenic pathways to accelerate tumor progression initiated by carcinogen, genetic mutation, or aging. Transcriptomic analysis uncovered an immediate expansion of the intestinal stem cell pool along with cell-autonomous Yki/Yap activation following disruption of Rab11a-mediated recycling endosomes. Intestinal tumors lacking Rab11a traffic exhibited marked elevation of nuclear Yap, upd3/IL6-Stat3, and amphiregulin-MAPK signaling, whereas suppression of Yki/Yap or upd3/IL6 reduced gut epithelial dysplasia and hyperplasia. Examination of Rab11a function in enteroids or cultured cell lines suggested that this endosome unit is required for suppression of the Yap pathway by Hippo kinases. Thus, recycling endosomes in mature epithelia constitute key tumor suppressors, loss of which accelerates carcinogenesis. SIGNIFICANCE: Recycling endosome traffic in mature epithelia constitutes a novel tumor suppressing mechanism.
Assuntos
Neoplasias Colorretais/metabolismo , Endossomos/metabolismo , Células Epiteliais/patologia , Proteínas rab de Ligação ao GTP/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Animais Geneticamente Modificados , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Células Epiteliais/metabolismo , Via de Sinalização Hippo , Humanos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/metabolismo , Células-Tronco/metabolismo , Células-Tronco/patologia , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Paneth cells are post-mitotic intestinal epithelial cells supporting the stem cell niche and mucosal immunity. Paneth cell pathologies are observed in various gastrointestinal diseases, but their plasticity and response to genomic and environmental challenges remain unclear. Using a knockin allele engineered at the mouse Lyz1 locus, we performed detailed Paneth cell-lineage tracing. Irradiation induced a subset of Paneth cells to proliferate and differentiate into villus epithelial cells. RNA sequencing (RNA-seq) revealed that Paneth cells sorted from irradiated mice acquired a stem cell-like transcriptome; when cultured in vitro, these individual Paneth cells formed organoids. Irradiation activated Notch signaling, and forced expression of Notch intracellular domain (NICD) in Paneth cells, but not Wnt/ß-catenin pathway activation, induced their dedifferentiation. This study documents Paneth cell plasticity, particularly their ability to participate in epithelial replenishment following stem cell loss, adding to a growing body of knowledge detailing the molecular pathways controlling injury-induced regeneration.