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
To mediate critical host-microbe interactions in the human small intestine, Paneth cells constitutively produce abundant levels of α-defensins and other antimicrobials. We report that the expression profile of these antimicrobials is dramatically askew in human small intestinal organoids (enteroids) as compared to that in paired tissue from which they are derived, with a reduction of α-defensins to nearly undetectable levels. Murine enteroids, however, recapitulate the expression profile of Paneth cell α-defensins seen in tissue. WNT/TCF signaling has been found to be instrumental in the regulation of α-defensins, yet in human enteroids exogenous stimulation of WNT signaling appears insufficient to rescue α-defensin expression. By stark contrast, forkhead box O (FOXO) inhibitor AS1842856 induced the expression of α-defensin mRNA in enteroids by >100,000-fold, restoring DEFA5 and DEFA6 to levels comparable to those found in primary human tissue. These results newly identify FOXO signaling as a pathway of biological and potentially therapeutic relevance for the regulation of human Paneth cell α-defensins in health and disease.
Assuntos
Anti-Infecciosos , alfa-Defensinas , Humanos , Animais , Camundongos , alfa-Defensinas/genética , alfa-Defensinas/farmacologia , alfa-Defensinas/metabolismo , Intestinos , Intestino Delgado/metabolismo , Celulas de Paneth/metabolismo , Anti-Infecciosos/metabolismo , Organoides/metabolismoRESUMO
In the mammalian intestine, flagellar motility can provide microbes competitive advantage, but also threatens the spatial segregation established by the host at the epithelial surface. Unlike microbicidal defensins, previous studies indicated that the protective activities of human α-defensin 6 (HD6), a peptide secreted by Paneth cells of the small intestine, resides in its remarkable ability to bind microbial surface proteins and self-assemble into protective fibers and nets. Given its ability to bind flagellin, we proposed that HD6 might be an effective inhibitor of bacterial motility. Here, we utilized advanced automated live cell fluorescence imaging to assess the effects of HD6 on actively swimming Salmonella enterica in real time. We found that HD6 was able to effectively restrict flagellar motility of individual bacteria. Flagellin-specific antibody, a classic inhibitor of flagellar motility that utilizes a mechanism of agglutination, lost its activity at low bacterial densities, whereas HD6 activity was not diminished. A single amino acid variant of HD6 that was able to bind flagellin, but not self-assemble, lost ability to inhibit flagellar motility. Together, these results suggest a specialized role of HD6 self-assembly into polymers in targeting and restricting flagellar motility.
Assuntos
Anti-Infecciosos , Celulas de Paneth , Animais , Humanos , Celulas de Paneth/metabolismo , Flagelina/metabolismo , Anti-Infecciosos/metabolismo , Bactérias/metabolismo , Flagelos/metabolismo , MamíferosRESUMO
It is unclear why disease occurs in only a small proportion of persons carrying common risk alleles of disease susceptibility genes. Here we demonstrate that an interaction between a specific virus infection and a mutation in the Crohn's disease susceptibility gene Atg16L1 induces intestinal pathologies in mice. This virus-plus-susceptibility gene interaction generated abnormalities in granule packaging and unique patterns of gene expression in Paneth cells. Further, the response to injury induced by the toxic substance dextran sodium sulfate was fundamentally altered to include pathologies resembling aspects of Crohn's disease. These pathologies triggered by virus-plus-susceptibility gene interaction were dependent on TNFalpha and IFNgamma and were prevented by treatment with broad spectrum antibiotics. Thus, we provide a specific example of how a virus-plus-susceptibility gene interaction can, in combination with additional environmental factors and commensal bacteria, determine the phenotype of hosts carrying common risk alleles for inflammatory disease.
Assuntos
Proteínas de Transporte/genética , Doença de Crohn/genética , Doença de Crohn/virologia , Predisposição Genética para Doença , Íleo/patologia , Norovirus , Animais , Proteínas Relacionadas à Autofagia , Doença de Crohn/patologia , Perfilação da Expressão Gênica , Humanos , Interferon gama/metabolismo , Camundongos , Celulas de Paneth/metabolismo , Celulas de Paneth/virologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
A decline in stem cell function impairs tissue regeneration during ageing, but the role of the stem-cell-supporting niche in ageing is not well understood. The small intestine is maintained by actively cycling intestinal stem cells that are regulated by the Paneth cell niche1,2. Here we show that the regenerative potential of human and mouse intestinal epithelium diminishes with age owing to defects in both stem cells and their niche. The functional decline was caused by a decrease in stemness-maintaining Wnt signalling due to production of Notum, an extracellular Wnt inhibitor, in aged Paneth cells. Mechanistically, high activity of mammalian target of rapamycin complex 1 (mTORC1) in aged Paneth cells inhibits activity of peroxisome proliferator activated receptor α (PPAR-α)3, and lowered PPAR-α activity increased Notum expression. Genetic targeting of Notum or Wnt supplementation restored function of aged intestinal organoids. Moreover, pharmacological inhibition of Notum in mice enhanced the regenerative capacity of aged stem cells and promoted recovery from chemotherapy-induced damage. Our results reveal a role of the stem cell niche in ageing and demonstrate that targeting of Notum can promote regeneration of aged tissues.
Assuntos
Envelhecimento , Senescência Celular , Esterases/metabolismo , Mucosa Intestinal/patologia , Celulas de Paneth/metabolismo , Regeneração , Envelhecimento/fisiologia , Animais , Senescência Celular/fisiologia , Esterases/antagonistas & inibidores , Esterases/biossíntese , Feminino , Humanos , Mucosa Intestinal/fisiologia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , PPAR alfa/metabolismo , Celulas de Paneth/patologia , Receptores Acoplados a Proteínas G/metabolismo , Nicho de Células-Tronco , Células-Tronco/patologia , Proteínas Wnt/antagonistas & inibidores , Via de Sinalização WntRESUMO
Paneth cells are intestinal epithelial cells that release antimicrobial peptides, such as α-defensin as part of host defense. Together with mesenchymal cells, Paneth cells provide niche factors for epithelial stem cell homeostasis. Here, we report two subtypes of murine Paneth cells, differentiated by their production and utilization of fucosyltransferase 2 (Fut2), which regulates α(1,2)fucosylation to create cohabitation niches for commensal bacteria and prevent invasion of the intestine by pathogenic bacteria. The majority of Fut2- Paneth cells were localized in the duodenum, whereas the majority of Fut2+ Paneth cells were in the ileum. Fut2+ Paneth cells showed higher granularity and structural complexity than did Fut2- Paneth cells, suggesting that Fut2+ Paneth cells are involved in host defense. Signaling by the commensal bacteria, together with interleukin 22 (IL-22), induced the development of Fut2+ Paneth cells. IL-22 was found to affect the α-defensin secretion system via modulation of Fut2 expression, and IL-17a was found to increase the production of α-defensin in the intestinal tract. Thus, these intestinal cytokines regulate the development and function of Fut2+ Paneth cells as part of gut defense.
Assuntos
Citocinas/metabolismo , Fucosiltransferases/metabolismo , Microbioma Gastrointestinal/fisiologia , Celulas de Paneth/metabolismo , Animais , Fucosiltransferases/genética , Íleo , Interleucina-17/metabolismo , Interleucinas/metabolismo , Camundongos , Simbiose , alfa-Defensinas/metabolismo , Interleucina 22 , Galactosídeo 2-alfa-L-FucosiltransferaseRESUMO
Adhesion G protein-coupled receptors (aGPCRs) feature large extracellular regions with modular domains that often resemble protein classes of various function. The pentraxin (PTX) domain, which is predicted by sequence homology within the extracellular region of four different aGPCR members, is well known to form pentamers and other oligomers. Oligomerization of GPCRs is frequently reported and mainly driven by interactions of the seven-transmembrane region and N or C termini. While the functional importance of dimers is well-established for some class C GPCRs, relatively little is known about aGPCR multimerization. Here, we showcase the example of ADGRG4, an orphan aGPCR that possesses a PTX-like domain at its very N-terminal tip, followed by an extremely long stalk containing serine-threonine repeats. Using X-ray crystallography and biophysical methods, we determined the structure of this unusual PTX-like domain and provide experimental evidence for a homodimer equilibrium of this domain which is Ca2+-independent and driven by intermolecular contacts that differ vastly from the known soluble PTXs. The formation of this dimer seems to be conserved in mammalian ADGRG4 indicating functional relevance. Our data alongside of theoretical considerations lead to the hypothesis that ADGRG4 acts as an in vivo sensor for shear forces in enterochromaffin and Paneth cells of the small intestine.
Assuntos
Fenômenos Biofísicos , Domínios Proteicos , Receptores Acoplados a Proteínas G , Transdução de Sinais , Animais , Mamíferos/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Células Enterocromafins/metabolismo , Celulas de Paneth/metabolismo , Cristalografia por Raios X , Fenômenos Biofísicos/fisiologia , Modelos Moleculares , Estrutura Terciária de Proteína , Dobramento de Proteína , Alinhamento de Sequência , Sequência de Aminoácidos , Células HEK293 , HumanosRESUMO
Although the role of the transcription factor NF-κB in intestinal inflammation and tumor formation has been investigated extensively, a physiological function of NF-κB in sustaining intestinal epithelial homeostasis beyond inflammation has not been demonstrated. Using NF-κB reporter mice, we detected strong NF-κB activity in Paneth cells, in '+4/+5' secretory progenitors and in scattered Lgr5+ crypt base columnar stem cells of small intestinal (SI) crypts. To examine NF-κB functions in SI epithelial self-renewal, mice or SI crypt organoids ('mini-guts') with ubiquitously suppressed NF-κB activity were used. We show that NF-κB activity is dispensable for maintaining SI epithelial proliferation, but is essential for ex vivo organoid growth. Furthermore, we demonstrate a dramatic reduction of Paneth cells in the absence of NF-κB activity, concomitant with a significant increase in goblet cells and immature intermediate cells. This indicates that NF-κB is required for proper Paneth versus goblet cell differentiation and for SI epithelial homeostasis, which occurs via regulation of Wnt signaling and Sox9 expression downstream of NF-κB. The current study thus presents evidence for an important role for NF-κB in intestinal epithelial self-renewal.
Assuntos
Células Caliciformes/citologia , Intestino Delgado/citologia , NF-kappa B/metabolismo , Celulas de Paneth/citologia , Animais , Diferenciação Celular , Autorrenovação Celular , Células Caliciformes/metabolismo , Homeostase , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Camundongos , NF-kappa B/genética , Organoides/citologia , Organoides/crescimento & desenvolvimento , Organoides/metabolismo , Celulas de Paneth/metabolismo , Fatores de Transcrição SOX9/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização WntRESUMO
Mouse atonal homolog 1 (Math1/Atoh1) is a basic helix-loop-helix transcription factor important for the differentiation of secretory cells within the intestinal epithelium. The analysis of Paneth depletion efficiency on Math1lox/loxVilCreERT2 (Math1ΔIEC) mice treatment with tamoxifen in the presence or absence of intestinal microbiota showed a failure on Paneth cell depletion in germ-free mice as compared with specific pathogen-free (SPF) mice. However, goblet cells were efficiently depleted in Math1ΔIEC germ-free mice. The gene expression of Math1 was significantly reduced in the ileum of germ-free Math1ΔIEC mice 5 days after tamoxifen injection as compared with germ-free control, but its protein expression was still detectable in the nuclei of epithelial cells in the crypts. Germ-free mice showed low proliferative ileal crypts and apoptotic cells that were mainly detected in the tip of the villus, consistent with a slow turnover rate of epithelial cells. Although Paneth cells were not depleted in germ-free Math1ΔIEC mice for the first 7 wk after the last tamoxifen injection, far already from the 5 days time-laps observed in SPF conditions, an incomplete depletion of Paneth cells was observed 14 wk after the last tamoxifen injection. Colonization of germ-free mice restored the phenotype observed in SPF mice, highlighting the regulatory role of gut microbes in our model. We conclude that absence of intestinal microbiota in Math1ΔIEC mice is associated with reduced epithelial cell renewal and delays the depletion of preexisting Paneth cells.NEW & NOTEWORTHY Cre-lox system is a powerful and widely used research tool developed to understand the specific role of genes. It allows to control the spatial and temporal expression of genes in experimental models. Several limitations including toxicity of Cre recombinase or incomplete excision of floxed loci have been reported in the past. To date, this is the first research study reporting that gut microbes also influence the expected phenotype of Paneth cell depletion in the genetically modified Math1lox/loxVilCreERT2 mouse model.
Assuntos
Microbioma Gastrointestinal , Celulas de Paneth , Camundongos , Animais , Celulas de Paneth/metabolismo , Mucosa Intestinal/metabolismo , Células Caliciformes/metabolismo , Tamoxifeno/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismoRESUMO
BACKGROUND: Mammalian intestinal epithelium constantly undergoes rapid self-renewal and regeneration sustained by intestinal stem cells (ISCs) within crypts. Inducible nitric oxide synthase (iNOS) is an important regulator in tissue homeostasis and inflammation. However, the functions of iNOS on ISCs have not been clarified. Here, we aimed to investigate the expression pattern of inducible nitric oxide synthase (iNOS) within crypts and explore its function in the homeostatic maintenance of the ISC niche. METHODS: Expression of iNOS was determined by tissue staining and qPCR. iNOS-/- and Lgr5 transgenic mice were used to explore the influence of iNOS ablation on ISC proliferation and differentiation. Enteroids were cultured to study the effect of iNOS on ISCs in vitro. Ileum samples from wild-type and iNOS-/- mice were collected for RNA-Seq to explore the molecular mechanisms by which iNOS regulates ISCs. RESULTS: iNOS was physiologically expressed in Paneth cells. Knockout of iNOS led to apparent morphological changes in the intestine, including a decrease in the small intestine length and in the heights of both villi and crypts. Knockout of iNOS decreased the number of Ki67+ or BrdU+ proliferative cells in crypts. Loss of iNOS increased the number of Olfm4+ ISCs but inhibited the differentiation and migration of Lgr5+ ISCs in vivo. iNOS depletion also inhibited enteroid formation and the budding efficiency of crypts in vitro. Moreover, iNOS deficiency altered gluconeogenesis and the adaptive immune response in the ileum transcriptome. CONCLUSION: Paneth cell-derived iNOS is required to maintain a healthy ISC niche, and Knockout of iNOS hinders ISC function in mice. Therefore, iNOS represents a potential target for the development of new drugs and other therapeutic interventions for intestinal disorders.
Assuntos
Celulas de Paneth , Nicho de Células-Tronco , Animais , Camundongos , Homeostase , Mucosa Intestinal/metabolismo , Intestinos , Mamíferos/metabolismo , Camundongos Knockout , Camundongos Transgênicos , Óxido Nítrico Sintase Tipo II/metabolismo , Celulas de Paneth/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Menopausal women often face long-term estrogen treatment. G protein-coupled estrogen receptor (GPER) expressed in intestinal crypt was activated by estrogen therapy, but it was unclear whether chronic GPER activation during menopause had an effect on intestinal stem cells (ISCs). We tested the effect of chronic GPER activation on ISCs of ovariectomized (OVX) mice by injection of the selective GPER agonist G-1 for 28 days, or G-1 stimulation of organoids derived from crypts of OVX mice. G-1 up-regulated crypt depth, the number of Ki67+, bromodeoxyuridine+ cells and Olfm4+ ISCs, and the expression of ISCs marker genes (Lgr5, Olfm4 and Axin2). G-1 administration promoted organoid growth, increased the number of EdU+ cells per organoid and protein expression of Cyclin D1 and cyclin B1 in organoids. After G-1 treatment in vivo or in vitro, Paneth cell-derived Wnt3, Wnt3 effector ß-catenin and Wnt target genes c-Myc and Cyclin D1 increased in ileum or organoids. Once blocking the secretion of Wnt3 from Paneth cells, the effects of G-1 on organoids growth, ISCs marker genes and Wnt/ß-catenin signaling were abolished. G-1 did not affect the number of Paneth cells in ex vivo organoids, while activated Mmp7/cryptdin program in Paneth cells, promoted their maturation, and increased the expression of lysozyme protein. G-1 pretreatment in OVX mice inhibited radiation-induced ISCs proliferation injury and enhanced the resistance of mice to intestinal injury. In conclusion, chronic GPER activation prompted the Wnt3 synthesis in Paneth cells, thus increased the proliferation of ISCs via activation of Wnt3/ß-catenin signaling in OVX mice.
Assuntos
Ciclina D1 , Celulas de Paneth , Camundongos , Feminino , Animais , Celulas de Paneth/metabolismo , Ciclina D1/metabolismo , beta Catenina/metabolismo , Íleo/metabolismo , Células-Tronco , Via de Sinalização Wnt , Proliferação de Células , Estrogênios/farmacologia , Estrogênios/metabolismo , Mucosa Intestinal/metabolismo , Proteína Wnt3/metabolismo , Proteína Wnt3/farmacologiaRESUMO
Intelectins (intestinal lectins) are highly conserved across chordate evolution and have been implicated in various human diseases, including Crohn's disease (CD). The human genome encodes two intelectin genes, intelectin-1 (ITLN1) and intelectin-2 (ITLN2). Other than its high sequence similarity with ITLN1, little is known about ITLN2. To address this void in knowledge, we report that ITLN2 exhibits discrete, yet notable differences from ITLN1 in primary structure, including a unique amino terminus, as well as changes in amino acid residues associated with the glycan-binding activity of ITLN1. We identified that ITLN2 is a highly abundant Paneth cell-specific product, which localizes to secretory granules, and is expressed as a multimeric protein in the small intestine. In surgical specimens of ileal CD, ITLN2 mRNA levels were reduced approximately five-fold compared to control specimens. The ileal expression of ITLN2 was unaffected by previously reported disease-associated variants in ITLN2 and CD-associated variants in neighboring ITLN1 as well as NOD2 and ATG16L1. ITLN2 mRNA expression was undetectable in control colon tissue; however, in both ulcerative colitis (UC) and colonic CD, metaplastic Paneth cells were found to express ITLN2. Together, the data reported establish the groundwork for understanding ITLN2 function(s) in the intestine, including its possible role in CD.
Assuntos
Doença de Crohn/metabolismo , Lectinas/metabolismo , Celulas de Paneth/metabolismo , Vesículas Secretórias/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Humanos , Lectinas/genética , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Adaptadora de Sinalização NOD2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Inflammatory bowel disease (IBD) has been attributed to aberrant mucosal immunity to the intestinal microbiota. The transcription factor XBP1, a key component of the endoplasmic reticulum (ER) stress response, is required for development and maintenance of secretory cells and linked to JNK activation. We hypothesized that a stressful environmental milieu in a rapidly proliferating tissue might instigate a proinflammatory response. We report that Xbp1 deletion in intestinal epithelial cells (IECs) results in spontaneous enteritis and increased susceptibility to induced colitis secondary to both Paneth cell dysfunction and an epithelium that is overly reactive to inducers of IBD such as bacterial products (flagellin) and TNFalpha. An association of XBP1 variants with both forms of human IBD (Crohn's disease and ulcerative colitis) was identified and replicated (rs35873774; p value 1.6 x 10(-5)) with novel, private hypomorphic variants identified as susceptibility factors. Hence, intestinal inflammation can originate solely from XBP1 abnormalities in IECs, thus linking cell-specific ER stress to the induction of organ-specific inflammation.
Assuntos
Proteínas de Ligação a DNA/imunologia , Retículo Endoplasmático/imunologia , Doenças Inflamatórias Intestinais/genética , Fatores de Transcrição/imunologia , Animais , Apoptose , Colite Ulcerativa/genética , Colite Ulcerativa/imunologia , Colite Ulcerativa/patologia , Doença de Crohn/genética , Doença de Crohn/imunologia , Doença de Crohn/patologia , Predisposição Genética para Doença , Humanos , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Listeria monocytogenes/imunologia , Camundongos , Camundongos Transgênicos , Celulas de Paneth/citologia , Celulas de Paneth/metabolismo , Fatores de Transcrição de Fator Regulador X , Proteína 1 de Ligação a X-BoxRESUMO
Intestinal epithelial cells absorb nutrients, respond to microbes, function as a barrier and help to coordinate immune responses. Here we report profiling of 53,193 individual epithelial cells from the small intestine and organoids of mice, which enabled the identification and characterization of previously unknown subtypes of intestinal epithelial cell and their gene signatures. We found unexpected diversity in hormone-secreting enteroendocrine cells and constructed the taxonomy of newly identified subtypes, and distinguished between two subtypes of tuft cell, one of which expresses the epithelial cytokine Tslp and the pan-immune marker CD45, which was not previously associated with non-haematopoietic cells. We also characterized the ways in which cell-intrinsic states and the proportions of different cell types respond to bacterial and helminth infections: Salmonella infection caused an increase in the abundance of Paneth cells and enterocytes, and broad activation of an antimicrobial program; Heligmosomoides polygyrus caused an increase in the abundance of goblet and tuft cells. Our survey highlights previously unidentified markers and programs, associates sensory molecules with cell types, and uncovers principles of gut homeostasis and response to pathogens.
Assuntos
Células Epiteliais/citologia , Epitélio/metabolismo , Intestino Delgado/citologia , Análise de Célula Única , Animais , Diferenciação Celular , Citocinas/metabolismo , Enterócitos/metabolismo , Células Epiteliais/metabolismo , Feminino , Perfilação da Expressão Gênica , Homeostase , Antígenos Comuns de Leucócito/metabolismo , Masculino , Camundongos , Organoides/citologia , Organoides/metabolismo , Celulas de Paneth/metabolismo , Transcrição Gênica , Linfopoietina do Estroma do TimoRESUMO
The small intestinal epithelium self-renews every four or five days. Intestinal stem cells (Lgr5+ crypt base columnar cells (CBCs)) sustain this renewal and reside between terminally differentiated Paneth cells at the bottom of the intestinal crypt. Whereas the signalling requirements for maintaining stem cell function and crypt homeostasis have been well studied, little is known about how metabolism contributes to epithelial homeostasis. Here we show that freshly isolated Lgr5+ CBCs and Paneth cells from the mouse small intestine display different metabolic programs. Compared to Paneth cells, Lgr5+ CBCs display high mitochondrial activity. Inhibition of mitochondrial activity in Lgr5+ CBCs or inhibition of glycolysis in Paneth cells strongly affects stem cell function, as indicated by impaired organoid formation. In addition, Paneth cells support stem cell function by providing lactate to sustain the enhanced mitochondrial oxidative phosphorylation in the Lgr5+ CBCs. Mechanistically, we show that oxidative phosphorylation stimulates p38 MAPK activation by mitochondrial reactive oxygen species signalling, thereby establishing the mature crypt phenotype. Together, our results reveal a critical role for the metabolic identity of Lgr5+ CBCs and Paneth cells in supporting optimal stem cell function, and we identify mitochondria and reactive oxygen species signalling as a driving force of cellular differentiation.
Assuntos
Autorrenovação Celular , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Células-Tronco/citologia , Animais , Diferenciação Celular , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Glicólise , Homeostase , Ácido Láctico/metabolismo , Camundongos , Mitocôndrias/metabolismo , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , Fosforilação Oxidativa , Celulas de Paneth/citologia , Celulas de Paneth/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Células-Tronco/fisiologia , Proteína Wnt3A/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Previous studies suggest that Paneth cells are involved in NEC development. Defensin alpha 6 (DEFA6) and guanylate cyclase activator 2A (GUCA2A) are selective protein markers of Paneth cells. The objective was to explore DEFA6 and GUCA2A expression in intestinal tissue samples from newborn infants with and without NEC. Tissue samples from histologically intact intestine were analyzed from 70 infants: 43 underwent bowel resection due to NEC and 27 controls were operated due to conditions such as intestinal atresia, dysmotility, aganglionosis, pseudo-obstruction or volvulus. Each tissue sample was immunohistochemically stained for DEFA6 and GUCA2A. Semi-automated digital image analysis was performed to determine protein expression. Clinical data and protein expressions were compared between the groups. DEFA6 expression was lower in the NEC group (p = 0.006). Low DEFA6 correlated with risk of developing NEC in a logistic regression analysis, independently of gestational age and birth weight (OR 0.843 [CI 0.732-0.971]; p = 0.018). GUCA2A expression did not differ between the two groups. CONCLUSION: Lower expression of DEFA6 together with intact GUCA2A expression indicates that NEC patients have well-defined Paneth cells but diminished defensin activity. Our results suggest that DEFA6 could be used as a biomarker for NEC. WHAT IS KNOWN: ⢠Previous studies of defensin activity in NEC have been inconsistent, showing that defensin levels may be increased or diminished in NEC. GUCA2A has to our knowledge never been studied in NEC. WHAT IS NEW: ⢠This study benchmarks two specific Paneth cell markers (DEFA6 and GUCA2A) and their activity in individuals with and without NEC. ⢠The key finding is that the NEC group had a lower DEFA6 expression compared to the Controls, while the expression of GUCA2A did not differ between the groups.
Assuntos
Enterocolite Necrosante , Doenças do Recém-Nascido , Lactente , Recém-Nascido , Humanos , Celulas de Paneth/metabolismo , Celulas de Paneth/patologia , Enterocolite Necrosante/diagnóstico , Peso ao Nascer , Idade Gestacional , Defensinas/metabolismoRESUMO
The proteolytic cleavage of histone tails, also termed histone clipping, has been described as a mechanism for permanent removal of post-translational modifications (PTMs) from histone proteins. Such activity has been ascribed to ensure regulatory function in key cellular processes such as differentiation, senescence and transcriptional control, for which different histone-specific proteases have been described. However, all these studies were exclusively performed using cell lines cultured in vitro and no clear evidence that histone clipping is regulated in vivo has been reported. Here we show that histone H3 N-terminal tails undergo extensive cleavage in the differentiated cells of the villi in mouse intestinal epithelium. Combining biochemical methods, 3D organoid cultures and in vivo approaches, we demonstrate that intestinal H3 clipping is the result of multiple proteolytic activities. We identified Trypsins and Cathepsin L as specific H3 tail proteases active in small intestinal differentiated cells and showed that their proteolytic activity is differentially affected by the PTM pattern of histone H3 tails. Together, our findings provide in vivo evidence of H3 tail proteolysis in mammalian tissues, directly linking H3 clipping to cell differentiation.
Assuntos
Enterócitos/metabolismo , Histonas/metabolismo , Intestino Delgado/citologia , Celulas de Paneth/metabolismo , Peptídeo Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional , Células-Tronco/metabolismo , Animais , Catepsina L/metabolismo , Diferenciação Celular , Homeostase , Mucosa Intestinal/citologia , Camundongos , Microvilosidades/ultraestrutura , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Organoides , Domínios Proteicos , Tripsina/metabolismoRESUMO
Paneth cells are antimicrobial peptide-secreting cells located at the base of the crypts of the small intestine. The proteome of Paneth cells is not well defined because of their coexistence with stem cells, making it difficult to culture Paneth cells alone in vitro. Using a simplified toluidine blue O method for staining mouse intestinal tissue, laser capture microdissection (LCM) to isolate cells from the crypt region, and surfactant-assisted one-pot protein digestion, we identified more than 1300 proteins from crypts equivalent to 18,000 cells. Compared with the proteomes of villi and smooth muscle regions, the crypt proteome is highly enriched in defensins, lysozymes, and other antimicrobial peptides that are characteristic of Paneth cells. The sensitivity of the LCM-based proteomics approach was also assessed using a smaller number of cell equivalent tissues: a comparable proteomic coverage can be achieved with 3600 cells. This work is the first proteomics study of intestinal tissue enriched with Paneth cells. The simplified workflow enables profiling of Paneth cell-associated pathological changes at the proteome level directly from frozen intestinal tissue. It may also be useful for proteomics studies of other spatially resolved cell types from other tissues.
Assuntos
Celulas de Paneth , Proteoma , Animais , Defensinas/metabolismo , Microdissecção e Captura a Laser/métodos , Camundongos , Celulas de Paneth/metabolismo , Proteoma/genética , Proteoma/metabolismo , Proteômica/métodos , Tensoativos , Cloreto de Tolônio/metabolismoRESUMO
BACKGROUND AND AIMS: Vitamin D exerts a regulatory role over mucosal immunity via the vitamin D receptor (VDR). Although Paneth cells and their products are known to regulate the commensal and pathogenic microbiota, the role that VDRs in Paneth cells play in these responses is unknown. METHODS: We identified the decreased intestinal VDR significantly correlated with reduction of an inflammatory bowel disease risk gene ATG16L1 and Paneth cell lysozymes in patients with Crohn's disease. We generated Paneth cell-specific VDR knockout (VDRΔPC) mice to investigate the molecular mechanisms. RESULTS: Lysozymes in the Paneth cells were significantly decreased in the VDRΔPC mice. Isolated VDRΔPC Paneth cells exhibited weakened inhibition of pathogenic bacterial growth and displayed reduced autophagic responses. VDRΔPC mice had significantly higher inflammation after Salmonella infections. VDRΔPC mice also showed high susceptibility to small intestinal injury induced by indomethacin, a nonsteroidal anti-inflammatory drug. Co-housing of VDRΔPC and VDRlox mice made the VDRΔPC less vulnerable to dextran sulfate sodium colitis, suggesting the transmission of protective bacterial from the VDRlox mice. Thus, a lack of VDR in Paneth cells leads to impaired antibacterial activities and consequently increased inflammatory responses. Genetically and environmentally regulated VDRs in the Paneth cells may set the threshold for the development of chronic inflammation, as observed in inflammatory bowel diseases. CONCLUSIONS: We provide new insights into the tissue-specific functions of VDRs in maintaining Paneth cell alertness to pathogens in intestinal disorders. Targeting the VDR affects multiple downstream events within Paneth cells that inhibit intestinal inflammation and establish host defense against enteropathogens.