RESUMEN
Bone marrow (BM)-resident hematopoietic stem and progenitor cells (HSPCs) are often activated following bacterial insults to replenish the host hemato-immune system, but how they integrate the associated tissue damage signals to initiate distal tissue repair is largely unknown. Here, we show that acute gut inflammation expands HSPCs in the BM and directs them to inflamed mesenteric lymph nodes through GM-CSFR activation for further expansion and potential differentiation into Ly6C+ /G+ myeloid cells specialized in gut tissue repair. We identified this process to be mediated by Bacteroides, a commensal gram-negative bacteria that activates innate immune signaling. These findings establish cross-organ communication between the BM and distant inflamed sites, whereby a certain subset of multipotent progenitors is specified to respond to imminent hematopoietic demands and to alleviate inflammatory symptoms.
Asunto(s)
Células Madre Hematopoyéticas , Inflamación , Humanos , Células Madre Hematopoyéticas/fisiología , Inflamación/patología , Diferenciación Celular , Transducción de Señal , Células Mieloides/patologíaRESUMEN
Bacterial infections can activate and mobilize hematopoietic stem and progenitor cells (HSPCs) from the bone marrow (BM) to the spleen, a process termed extramedullary hematopoiesis (EMH). Recent studies suggest that commensal bacteria regulate not only the host immune system but also hematopoietic homeostasis. However, the impact of gut microbes on hematopoietic pathology remains unclear. Here, we find that systemic single injections of Akkermansia muciniphila (A. m.), a mucin-degrading bacterium, rapidly activate BM myelopoiesis and slow but long-lasting hepato-splenomegaly, characterized by the expansion and differentiation of functional HSPCs, which we term delayed EMH. Mechanistically, delayed EMH triggered by A. m. is mediated entirely by the MYD88/TRIF innate immune signaling pathway, which persistently stimulates splenic myeloid cells to secrete interleukin (IL)-1α, and in turn, activates IL-1 receptor (IL-1R)-expressing splenic HSPCs. Genetic deletion of Toll-like receptor-2 and -4 (TLR2/4) or IL-1α partially diminishes A. m.-induced delayed EMH, while inhibition of both pathways alleviates splenomegaly and EMH. Our results demonstrate that cooperative IL-1R- and TLR-mediated signals regulate commensal bacteria-driven EMH, which might be relevant for certain autoimmune disorders.
Asunto(s)
Hematopoyesis Extramedular , Humanos , Hematopoyesis Extramedular/genética , Esplenomegalia/metabolismo , Médula Ósea , Células Madre Hematopoyéticas/metabolismo , HematopoyesisRESUMEN
How commensal microbiota contributes to immune cell homeostasis at barrier surfaces is poorly understood. Lamina propria (LP) T helper 17 (Th17) cells participate in mucosal protection and are induced by commensal segmented filamentous bacteria (SFB). Here we show that MHCII-dependent antigen presentation of SFB antigens by intestinal dendritic cells (DCs) is crucial for Th17 cell induction. Expression of MHCII on CD11c(+) cells was necessary and sufficient for SFB-induced Th17 cell differentiation. Most SFB-induced Th17 cells recognized SFB in an MHCII-dependent manner. SFB primed and induced Th17 cells locally in the LP and Th17 cell induction occurred normally in mice lacking secondary lymphoid organs. The importance of other innate cells was unveiled by the finding that MHCII deficiency in group 3 innate lymphoid cells (ILCs) resulted in an increase in SFB-independent Th17 cell differentiation. Our results outline the complex role of DCs and ILCs in the regulation of intestinal Th17 cell homeostasis.
Asunto(s)
Antígenos Bacterianos/inmunología , Infecciones por Clostridium/inmunología , Clostridium/inmunología , Células Dendríticas/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Intestinos/inmunología , Linfocitos/inmunología , Células Th17/inmunología , Animales , Presentación de Antígeno , Diferenciación Celular , Células Cultivadas , Células Dendríticas/microbiología , Antígenos de Histocompatibilidad Clase II/genética , Intestinos/microbiología , Activación de Linfocitos , Ratones , Ratones Noqueados , Ratones Transgénicos , Microbiota/inmunología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismoRESUMEN
Gut commensal microbes shape the mucosal immune system by regulating the differentiation and expansion of several types of T cell. Clostridia, a dominant class of commensal microbe, can induce colonic regulatory T (Treg) cells, which have a central role in the suppression of inflammatory and allergic responses. However, the molecular mechanisms by which commensal microbes induce colonic Treg cells have been unclear. Here we show that a large bowel microbial fermentation product, butyrate, induces the differentiation of colonic Treg cells in mice. A comparative NMR-based metabolome analysis suggests that the luminal concentrations of short-chain fatty acids positively correlates with the number of Treg cells in the colon. Among short-chain fatty acids, butyrate induced the differentiation of Treg cells in vitro and in vivo, and ameliorated the development of colitis induced by adoptive transfer of CD4(+) CD45RB(hi) T cells in Rag1(-/-) mice. Treatment of naive T cells under the Treg-cell-polarizing conditions with butyrate enhanced histone H3 acetylation in the promoter and conserved non-coding sequence regions of the Foxp3 locus, suggesting a possible mechanism for how microbial-derived butyrate regulates the differentiation of Treg cells. Our findings provide new insight into the mechanisms by which host-microbe interactions establish immunological homeostasis in the gut.
Asunto(s)
Butiratos/metabolismo , Diferenciación Celular , Colon/inmunología , Colon/microbiología , Fermentación , Simbiosis , Linfocitos T Reguladores/citología , Acetilación/efectos de los fármacos , Traslado Adoptivo , Animales , Butiratos/análisis , Butiratos/farmacología , Diferenciación Celular/efectos de los fármacos , Colitis/tratamiento farmacológico , Colitis/patología , Colon/citología , Colon/metabolismo , Secuencia Conservada , Femenino , Factores de Transcripción Forkhead/genética , Vida Libre de Gérmenes , Histonas/metabolismo , Homeostasis/efectos de los fármacos , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Recuento de Linfocitos , Espectroscopía de Resonancia Magnética , Masculino , Metaboloma , Ratones , Regiones Promotoras Genéticas/efectos de los fármacos , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunologíaRESUMEN
Bacterial access to the gut immune system is a crucial process to promote host immune responses. The probiotic L-92 strain of Lactobacillus acidophilus exerts anti-allergic immunomodulatory effects upon oral administration in mice. Here, we show that microfold cells (M cells) are responsible for L-92 internalization for evoking L-92-mediated immune responses. L-92 specifically bound to uromodulin, a glycosylphosphatidylinositol-anchored protein expressed exclusively on M cells among intestinal epithelial cells. Internalization of L-92 into M cells was significantly reduced in uromodulin-deficient (Umod-/-) mice compared to Umod+/+ mice. Furthermore, the binding of L-92 to uromodulin was significantly decreased after removal of surface layer protein A (SlpA) from the bacteria. Our study thus revealed a crucial role of uromodulin on the M-cell surface for the uptake of SlpA-positive lactic acid bacteria into M cells, possibly leading to subsequent delivery of the bacteria to dendritic cells closely associated with M cells for immunomodulation. Our study also shed light on the possibility that SlpA and uromodulin could be used as vehicle and target, respectively, for efficient mucosal vaccine delivery.
Asunto(s)
Proteínas Bacterianas/metabolismo , Células Dendríticas/inmunología , Mucosa Intestinal/metabolismo , Lactobacillus acidophilus/fisiología , Uromodulina/metabolismo , Animales , Células Cultivadas , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de Órganos , Probióticos , Unión Proteica , Transcriptoma , Uromodulina/genéticaRESUMEN
Nasopharynx-associated lymphoid tissue (NALT) is one of the major constituents of the mucosa-associated lymphoid tissue (MALT), and has the ability to induce antigen-specific immune responses. However, the molecular mechanisms responsible for antigen uptake from the nasal cavity into the NALT remain largely unknown. Immunohistochemical analysis showed that CCL9 and CCL20 were co-localized with glycoprotein 2 (GP2) in the epithelium covering NALT, suggesting the existence of M cells in NALT. In analogy with the reduced number of Peyer's patch M cells in CCR6-deficient mice, the number of NALT M cells was drastically decreased in CCR6-deficient mice compared with the wild-type mice. Translocation of nasally administered Salmonella enterica serovar Typhimurium into NALT via NALT M cells was impaired in CCR6-deficient mice, whereas S. Typhimurium demonstrated consistent co-localization with NALT M cells in wild-type mice. When wild-type mice were nasally administered with an attenuated vaccine strain of S. Typhimurium, the mice were protected from a subsequent challenge with wild-type S. Typhimurium. Antigen-specific fecal and nasal IgA was detected after nasal immunization with the attenuated vaccine strain of S. Typhimurium only in wild-type mice but not in CCR6-deficient mice. Taken together, these observations demonstrate that NALT M cells are important as a first line of defense against infection by enabling activation of the common mucosal immune system (CMIS).
Asunto(s)
Células Epiteliales/inmunología , Inmunidad Mucosa/inmunología , Tejido Linfoide/inmunología , Nasofaringe/inmunología , Animales , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
Intrinsic Notch signaling in intestinal epithelial cells restricts secretory cell differentiation. In gut-associated lymphoid tissue (GALT), stromal cells located beneath the follicle-associated epithelium (FAE) abundantly express the Notch ligand delta-like 1 (Dll1). Here, we show that mice lacking Rbpj-a gene encoding a transcription factor implicated in Notch signaling-in intestinal epithelial cells have defective GALT maturation. This defect can be attributed to the expansion of goblet cells, which leads to the down-regulation of CCL20 in FAE. These data demonstrate that epithelial Notch signaling maintained by stromal cells contributes to the full maturation of GALT by restricting secretory cell differentiation in FAE.
Asunto(s)
Células Epiteliales/citología , Células Epiteliales/metabolismo , Tejido Linfoide/citología , Tejido Linfoide/metabolismo , Receptores Notch/metabolismo , Células del Estroma/citología , Animales , Diferenciación Celular/fisiología , Técnicas In Vitro , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Ratones , Transducción de Señal/fisiología , Células del Estroma/metabolismoRESUMEN
The mucosal immune system forms the largest part of the entire immune system, containing about three-quarters of all lymphocytes and producing grams of secretory IgA daily to protect the mucosal surface from pathogens. To evoke the mucosal immune response, antigens on the mucosal surface must be transported across the epithelial barrier into organized lymphoid structures such as Peyer's patches. This function, called antigen transcytosis, is mediated by specialized epithelial M cells. The molecular mechanisms promoting this antigen uptake, however, are largely unknown. Here we report that glycoprotein 2 (GP2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for mucosal antigens. Recombinant GP2 protein selectively bound a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane. Consistently, these bacteria were colocalized with endogenous GP2 on the apical plasma membrane as well as in cytoplasmic vesicles in M cells. Moreover, deficiency of bacterial FimH or host GP2 led to defects in transcytosis of type-I-piliated bacteria through M cells, resulting in an attenuation of antigen-specific immune responses in Peyer's patches. GP2 is therefore a previously unrecognized transcytotic receptor on M cells for type-I-piliated bacteria and is a prerequisite for the mucosal immune response to these bacteria. Given that M cells are considered a promising target for oral vaccination against various infectious diseases, the GP2-dependent transcytotic pathway could provide a new target for the development of M-cell-targeted mucosal vaccines.
Asunto(s)
Adhesinas de Escherichia coli/metabolismo , Antígenos Bacterianos/metabolismo , Células Epiteliales/inmunología , Proteínas Fimbrias/metabolismo , Inmunidad Mucosa/inmunología , Glicoproteínas de Membrana/metabolismo , Ganglios Linfáticos Agregados/citología , Adhesinas de Escherichia coli/genética , Adhesinas de Escherichia coli/inmunología , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Línea Celular , Células Epiteliales/metabolismo , Escherichia coli/inmunología , Escherichia coli/metabolismo , Proteínas Fimbrias/genética , Proteínas Fimbrias/inmunología , Proteínas Ligadas a GPI , Glicoproteínas , Células HeLa , Humanos , Intestinos/citología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ganglios Linfáticos Agregados/inmunología , Salmonella typhimurium/genética , Salmonella typhimurium/inmunología , Salmonella typhimurium/metabolismo , Especificidad por SustratoRESUMEN
Microfold (M) cells are intestinal epithelial cells specialized for sampling and transport of luminal antigens to gut-associated lymphoid tissue for initiation of both mucosal and systemic immune responses. Therefore, M-cell targeted vaccination has the potential to be a better immunization strategy. Glycoprotein 2 (GP2), an antigen uptake receptor for FimH(+) bacteria on M cells, can be a good target for this purpose. Aptamers are oligonucleotides that bind to a variety of target molecules with high specificity and affinity. Together with its low toxic feature, aptamers serves as a tool of molecular-targeted delivery. In this study, we used Systematic Evolution of Ligands by EXponential enrichment (SELEX) to isolate aptamers specific to murine GP2 (mGP2). After ten rounds of SELEX, eleven different aptamer sequences were selected. Among them, the most frequently appeared sequence (~60%) were aptamer NO. 1 (Apt1), and the second most (~7%) were aptamer NO. 5 (Apt5). In vitro binding experiment confirmed that only Apt1 and Apt5 specifically bound to mGP2 among eleven aptamers initially selected. Apt1 showed the strongest affinity with mGP2, with the Kd value of 110±2.6 nM evaluated by BIACORE. Binding assays with mutants of Apt1 suggest that, in addition to the loop structure, the nucleotide sequence, AAAUA, in the loop is important for binding to mGP2. Furthermore, this aptamer was able to bind to mGP2 expressed on the cell surface. These results suggest that this mGP2-specific aptamer could serve as a valuable tool for testing M-cell-targeted vaccine delivery in the murine model system.
Asunto(s)
Aptámeros de Nucleótidos/metabolismo , Proteínas Ligadas a GPI/metabolismo , Técnica SELEX de Producción de Aptámeros , Animales , Aptámeros de Nucleótidos/genética , Secuencia de Bases , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Ratones , Especificidad por SustratoRESUMEN
Brucella abortus is a Gram-negative bacterium causing brucellosis. Although B. abortus is known to infect via the oral route, the entry site in the gastrointestinal tract has been unclear. We found that B. abortus was selectively internalized by microfold cells (M cells), a subset of epithelial cells specialized for mucosal Ag uptake. During this process, colocalization of cellular prion protein (PrP(C)) and B. abortus was evident on the apical surface as well as in subapical vacuolar structures in M cells. Internalization of B. abortus by M cells of PrP(C)-deficient (Prnp(-/-)) mice was greatly reduced compared with that in wild-type mice. Furthermore, an oral infection study revealed that translocation of B. abortus into the Peyer's patch was significantly lower in Prnp(-/-) than in wild-type mice. These observations suggest that orally infected B. abortus invades the host through M cells by using PrP(C) on the apical surface of M cells as an uptake receptor.
Asunto(s)
Brucella abortus/metabolismo , Brucelosis/metabolismo , Mucosa Intestinal/metabolismo , Proteínas PrPC/metabolismo , Animales , Brucella abortus/patogenicidad , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ganglios Linfáticos Agregados/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Receptor activator of nuclear factor kappa-B ligand (RANKL) expression was examined during the development of mouse fetal peripheral lymphoid organs. A shift in the expression pattern was detected during the transition from lymphoid tissue inducer (LTi) cells to lymphoid tissue organizer (LTo) cells in the lymph node (LN) anlagen but not in the Peyer's patch anlagen. In order to understand the functional impact of these changes in the fetal expression of RANKL, the RANKL function was blocked by a blocking antibody. Excess anti-RANKL antibody was administered to pregnant mice between 13.5 and 16.5 dpc and was found to completely block LN anlagen development, suggesting that RANKL function during this period is critical for LN development. In addition, small amounts of anti-RANKL antibodies were injected directly into the amniotic space at 13.5 dpc, resulting in perturbed B-cell follicle formation and high endothelial venule differentiation after birth. These results suggest that RANKL expression on LTi cells during the early phase of LN development is critical for the development LN microarchitecture.
Asunto(s)
Embrión de Mamíferos/metabolismo , Perfilación de la Expresión Génica , Ganglios Linfáticos/metabolismo , Ligando RANK/genética , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Microambiente Celular/efectos de los fármacos , Microambiente Celular/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Femenino , Inmunohistoquímica , Ganglios Linfáticos/citología , Ganglios Linfáticos/embriología , Tejido Linfoide/citología , Tejido Linfoide/embriología , Tejido Linfoide/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Organogénesis/efectos de los fármacos , Organogénesis/genética , Ganglios Linfáticos Agregados/citología , Ganglios Linfáticos Agregados/embriología , Ganglios Linfáticos Agregados/metabolismo , Embarazo , Ligando RANK/inmunología , Ligando RANK/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: It is estimated that by 2040 there will be 1,017,712 new cases of prostate cancer worldwide. Androgen deprivation therapy (ADT) is widely used as a treatment option for all disease stages. ADT, and the resulting decline in androgen levels, may indirectly affect gut microbiota. Factors affecting gut microbiota are wide-ranging; however, literature is scarce on the effects of ADT on gut microbiota and metabolome profiles in patients with prostate cancer. METHODS: To study the changes of gut microbiome by ADT, this 24-week observational study investigated the relationship between testosterone levels and changes in gut microbiota in Japanese patients with prostate cancer undergoing ADT. Sequential faecal samples were collected 1 and 2 weeks before ADT, and 1, 4, 12, and 24 weeks after ADT. Blood samples were collected at almost the same times. Bacterial 16 S rRNA gene-based microbiome analyses and capillary electrophoresis-time-of-flight mass spectrometry-based metabolome analyses were performed. RESULTS: In total, 23 patients completed the study. The α- and ß-diversity of gut microbiota decreased significantly at 24 weeks after ADT (p = 0.017, p < 0.001, respectively). Relative abundances of Proteobacteria, Gammaproteobacteria, Pseudomonadales, Pseudomonas, and concentrations of urea, lactate, butyrate, 2-hydroxyisobutyrate and S-adenosylmethionine changed significantly after ADT (p < 0.05). There was a significant positive correlation between the abundance of Proteobacteria, a known indicator of dysbiosis, and the concentration of lactate (R = 0.49, p < 0.01). CONCLUSIONS: The decline in testosterone levels resulted in detrimental changes in gut microbiota. This dysbiosis may contribute to an increase in frailty and an increased risk of adverse outcomes in patients with prostate cancer.
Asunto(s)
Neoplasias de la Próstata , Masculino , Humanos , Antagonistas de Andrógenos/efectos adversos , Andrógenos , Disbiosis/inducido químicamente , Testosterona , LactatosRESUMEN
The homologs EpCAM and TROP2, which both interact with claudin-1 and claudin-7, are frequently coexpressed in epithelia including skin. Intestine uniquely expresses high levels of EpCAM but not TROP2. We previously identified EpCAM as a substrate of the membrane-anchored protease matriptase and linked HAI-2, matriptase, EpCAM and claudin-7 in a pathway that is pivotal for intestinal epithelial cells (IEC) homeostasis. Herein, we reveal that TROP2 is also a matriptase substrate. Matriptase cleaved TROP2 when purified recombinant proteins were mixed in vitro. TROP2, like EpCAM, was also cleaved after co-transfection of matriptase in 293T cells. Neither EpCAM nor TROP2 cleavage was promoted by protease-disabled matriptase or matriptase that harbored the ichthyosis-associated G827R mutation. We confirmed that EpCAM and TROP2 are both expressed in skin and detected cleavage of these proteins in human keratinocytes (HaCaT cells) after the physiologic inhibition of matriptase by HAI proteins was relieved by siRNA knockdown. Knockdown of EpCAM or TROP2 individually had only small effects on claudin-1 and claudin-7 levels, whereas elimination of both markedly diminished claudin levels. HAI-1 knockdown promoted EpCAM and TROP2 cleavage accompanied by reductions in claudins, whereas HAI-2 knockdown had little impact. Double knockdown of HAI-1 and HAI-2 induced nearly complete cleavage of EpCAM and TROP2 and drastic reductions of claudins. These effects were eliminated by concurrent matriptase knockdown. Decreases in claudin levels were also diminished by the lysosomal inhibitor chloroquine and cleaved EpCAM/TROP2 fragments accumulated preferentially. We demonstrate that TROP2 and EpCAM exhibit redundancies with regard to regulation of claudin metabolism and that an HAI, matriptase, EpCAM and claudin pathway analogous to what we described in IECs exists in keratinocytes. This study may offer insights into the mechanistic basis for matriptase dysregulation-induced ichthyosis.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Moléculas de Adhesión Celular/metabolismo , Claudinas/metabolismo , Molécula de Adhesión Celular Epitelial/metabolismo , Queratinocitos/metabolismo , Serina Endopeptidasas/metabolismo , Animales , Células HaCaT , Humanos , Intestinos/fisiología , Lisosomas/metabolismo , Ratones Endogámicos C57BL , Proteínas Mutantes/metabolismo , Estabilidad Proteica , Proteolisis , Piel/metabolismoRESUMEN
TROP1 (EpCAM) and TROP2 are homologous cell surface proteins that are widely expressed, and often co-expressed, in developing and adult epithelia. Various functions have been ascribed to EpCAM and TROP2, but responsible mechanisms are incompletely characterized and functional equivalence has not been examined. Adult intestinal epithelial cells (IEC) express high levels of EpCAM, while TROP2 is not expressed. EpCAM deficiency causes congenital tufting enteropathy (CTE) in humans and a corresponding lethal condition in mice. We expressed TROP2 and EpCAM in the IEC of EpCAM-deficient mice utilizing a villin promoter to assess EpCAM and TROP2 function. Expression of EpCAM or TROP2 in the IEC of EpCAM knockout mice prevented CTE. TROP2 rescue (T2R) mice were smaller than controls, while EpCAM rescue (EpR) mice were not. Abnormalities were observed in the diameters and histology of T2R small intestine, and Paneth and stem cell markers were decreased. T2R mice also exhibited enlarged mesenteric lymph nodes, enhanced permeability to 4 kDa FITC-dextran and increased sensitivity to detergent-induced colitis, consistent with compromised barrier function. Studies of IEC organoids and spheroids revealed that stem cell function was also compromised in T2R mice. We conclude that EpCAM and TROP2 exhibit functional redundancy, but they are not equivalent.
Asunto(s)
Antígenos de Neoplasias/fisiología , Moléculas de Adhesión Celular/fisiología , Diarrea Infantil/genética , Molécula de Adhesión Celular Epitelial/fisiología , Células Epiteliales/metabolismo , Síndromes de Malabsorción/genética , Animales , Células HEK293 , Humanos , Mucosa Intestinal/citología , Ratones Endogámicos C57BL , Ratones Noqueados , Regiones Promotoras GenéticasRESUMEN
Epidermal-specific deletion of the homeobox transcription regulator DLX3 disrupts keratinocyte differentiation and results in an IL-17-linked psoriasis-like skin inflammation. To identify the epidermal initiating signals produced by DLX3-null keratinocytes, we performed acute deletion of DLX3 in adult epidermis using a tamoxifen-inducible Krt14-cre/ERT system. K14CreERT;DLX3fl/fl skin exhibited dysregulated expression of differentiation-associated genes, upregulation of proinflammatory cytokines, and accumulation of Langerhans cells and macrophages within 3 days of tamoxifen-induced DLX3 ablation. We also observed increased accumulation of IL-17A-secreting Vγ4 γδ T cells and heightened levels of IL-17 and IL-36 family of cytokines starting 1 week after DLX3 deletion. Interestingly, transcriptome profiling of K14CreERT;DLX3fl/fl epidermis at 3 days identified activated STAT3 as a transcriptional regulator and revealed differential expression of STAT3 signaling-related genes. Furthermore, activation of STAT3 was strongly increased in K14CreERT;DLX3fl/fl skin, and topical treatment with an inhibitor of STAT3 activation attenuated the immune phenotype. RNA-seq analysis of vehicle and STAT3 inhibitor treated K14CreERT;DLX3fl/fl skin identified differentially expressed genes associated with inhibition of leukocyte infiltration. Collectively, our results show that DLX3 is a critical regulator of STAT3 signaling network that maintains skin homeostasis.
Asunto(s)
Proteínas de Homeodominio/fisiología , Homeostasis , Queratinocitos/fisiología , Factor de Transcripción STAT3/fisiología , Transducción de Señal/fisiología , Piel/inmunología , Factores de Transcripción/fisiología , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Dermatitis/etiología , Ratones , Factor de Transcripción STAT3/antagonistas & inhibidoresRESUMEN
Langerhans cells (LCs) induce type 2 antibodies reactive with protein antigens that are applied to murine skin in the absence of adjuvant after extending their dendrites through tight junctions to acquire antigens and migrating to regional lymph nodes. In response to contact sensitizers, epithelial cell adhesion molecule (EpCAM) on LCs promotes LC dendrite mobility and LC migration. In epithelial cells, EpCAM regulates expression and distribution of selected tight junctions-associated claudins. To determine if EpCAM regulates claudins in LC and immune responses to externally applied proteins, we studied conditional knockout mice with EpCAM-deficient LCs. Although LC claudin-1 levels were dramatically reduced in the absence of EpCAM, conditional knockout mice with EpCAM-deficient LCs and control LC dendrites docked with epidermal tight junctions with equal efficiencies and ingested surface proteins. Topical immunization of conditional knockout mice with EpCAM-deficient LCs with ovalbumin led to increased induction of type 2 Ova-specific antibodies and enhanced proliferation of ovalbumin-reactive T cells associated with increased accumulation of LCs in lymph nodes. These results suggest that, in the absence of strong adjuvants, EpCAM-deficient LCs exhibit increased migration to regional lymph nodes. EpCAM appears to differentially regulate LC mobility/migration in the setting of limited inflammation as compared with the intense inflammation triggered by contact sensitizers.
Asunto(s)
Antígenos/inmunología , Células Epidérmicas , Molécula de Adhesión Celular Epitelial/metabolismo , Regulación de la Expresión Génica , Células de Langerhans/metabolismo , Uniones Estrechas/metabolismo , Animales , Movimiento Celular , Claudina-1/metabolismo , Dendritas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Epidermis/inmunología , Femenino , Inmunización , Células de Langerhans/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía FluorescenteRESUMEN
M cells in the follicle-associated epithelium (FAE) of Peyer's patches (PPs) serve as a main portal for external antigens and function as a sentinel in mucosal immune responses. The scarcity of these cells has hampered identification of M cell-specific molecules. Recent efforts have begun to provide insight into antigen transcytosis and differentiation of M cells; however, the molecular mechanisms underlying these processes are not fully elucidated. Small non-coding RNAs including microRNA (miRNA) have been reported to regulate gene expression and control various biological processes such as cellular differentiation and function. To evaluate the expression of miRNAs in FAE, including M cells, we previously performed microarray analysis comparing intestinal villous epithelium (VE) and PP FAE. Here we confirmed FAE specific miRNA expression levels by quantitative PCR. To gain insight into miRNA function, we generated mice with intestinal epithelial cell-specific deletion of Dicer1 (DicerΔIEC) and analyzed intestinal phenotypes, including M-cell differentiation, morphology and function. DicerΔIEC mice had a marked decrease in M cells compared to control floxed Dicer mice, suggesting an essential role of miRNAs in maturation of these cells. Furthermore, transmission electron microscopic analysis revealed that depletion of miRNA caused the loss of endosomal structures in M cells. In addition, antigen uptake by M cells was impaired in DicerΔIEC mice. These results suggest that miRNAs play a significant role in M cell differentiation and help secure mucosal immune homeostasis.
Asunto(s)
Homeostasis/inmunología , Inmunidad Mucosa/inmunología , Mucosa Intestinal/inmunología , MicroARNs/inmunología , Animales , Diferenciación Celular/inmunología , Células Epiteliales/inmunología , Regulación de la Expresión Génica/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Transcitosis/inmunologíaRESUMEN
M cells in follicle-associated epithelium (FAE) covering intestinal lymphoid follicles serve as a portal of entry for particulate antigens (Kanaya and Ohno, 2014 [1]). Despite their biological significance, molecular mechanisms that govern M-cell differentiation and function have not been fully elucidated. MicroRNAs (miRNAs) have a role to control host gene expression profiles that modulate cellular physiology and characteristic. Many studies have shown that miRNAs regulate diverse biological processes including developmental timing, differentiation and growth control of cells and tissues (Ivey and Srivastava, 2010 [2]). miRNAs are also relevant to differentiation and function of intestinal epithelium (McKenna et al., 2010 [3]; Runtsch et al., 2014 [4]). Expression profiles and functions of miRNAs in the intestinal epithelium have been examined in jejunal and colonic mucosa [3]. In contrast, those in FAE remain uncharacterized. To address this deficiency, we isolated Peyer's Patch (PP) FAE and villous epithelium (VE) surrounding the FAE, and compared the miRNA expression profiles of FAE and VE by microarray analysis. This revealed that 43 miRNAs were up-regulated, whereas 9 miRNAs were down-regulated, in FAE compared to VE. A unique pattern of miRNA expression by FAE may reflect important diversity in cellular phenotypes and/or functional features of FAE. All microarray data has been deposited at GEO under accession number GSE46264.
RESUMEN
Segmented filamentous bacteria (SFB) are Gram-positive, anaerobic, spore-forming commensals that reside in the gut of many animal species. Described more than forty years ago, SFB have recently gained interest due to their unique ability to modulate the host immune system through induction of IgA and Th17 cells. Here, we describe a collection of methods to detect and quantify SFB and SFB adhesion in intestinal mucosa, as well as SFB-specific CD4 T cells in the lamina propria. In addition, we describe methods for purification of SFB from fecal material of SFB-monoassociated gnotobiotic mice. Using these methods we examine the kinetics of SFB colonization and Th17 cell induction. We also show that SFB colonize unevenly the intestinal mucosa and that SFB adherence occurs predominantly in the terminal ileum and correlates with an increased proportion of SFB-specific Th17 cells.
Asunto(s)
Infecciones por Bacterias Grampositivas/inmunología , Bacterias Grampositivas Formadoras de Endosporas/inmunología , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Células Th17/inmunología , Animales , Adhesión Bacteriana/inmunología , Heces/microbiología , Ratones , Ratones Endogámicos C57BL , Microbiota/inmunología , SimbiosisRESUMEN
A minor population of M cells within the follicle-associated epithelium (FAE) of intestinal Peyer's patches (PPs) serves as a major portal for entry of exogenous antigens. Characterization of the mammalian M cells, including identification of M-cell surface molecules used for bacterial uptake, has been hampered by their relative rarity. In contrast, M cells constitute virtually all of the FAE cells in the avian bursa of Fabricius. We therefore performed comparative gene expression profiling of chicken and murine FAE to identify commonly expressed genes by M cells in both species. The comprehensive transcriptome analysis revealed that 28 genes were commonly up-regulated in FAE from both species. In situ hybridization revealed that annexin A10 (Anxa10) mRNA was scattered in FAE, and co-localized with Ulex europaeus agglutinin-1 binding to M cells. Whole-mount immunostaining also revealed that cellular prion protein (PrP(C)) was expressed on the luminal side of the apical plasma membrane of M cells, and co-localized with grycoprotein 2 that recognizes only M cells in murine PP. Our findings identify new M-cell-specific molecules through using comprehensive transcriptome analysis. These conserved molecules in M cells of mice and chickens may play essential roles in M-cell function and/or differentiation.