RESUMO
Apelin (APL), an endogenous ligand for APJ, has been reported to be upregulated in a murine model of acute colitis induced by sodium dextran sulfate, as well as inflammatory bowel diseases (IBD) in humans. However, the mechanisms and functions of APL/APJ axis in the pathogenesis of IBD are unclear. We herein analyzed CD4+ T cells to determine the functions of APL in a murine model of chronic colitis induced in Rag deficient mice (Rag-/-). In colonic tissues of wild-type mice (WT), we found that APL was expressed especially in the lamina propria lymphocytes, where CD4+ T cells are dominant, rather than the epithelial cells. Unexpectedly, the APL expression was rather downregulated in the colonic tissue of the chronic colitis group compared to the control groups (Rag-/- before colitis induction and WT). The APL expression was downregulated when naïve T cells were differentiated into effecter T cells. A lack of APL resulted in decreased naïve T cells and increased effecter T cells in secondary lymphoid organs. A synthetic APL peptide, [Pyr1]-APL-13, increased IL-10 and decreased IFN-γ productions by effecter T cells. Administration of [Pyr1]-APL-13 improved survival rate in association with lessened colitis severity and decreased pro-inflammatory cytokine production. This is the first report showing immunological function of APL specifically on T cells, and these results indicate that APL/APJ axis may be a novel therapeutic target for IBD.
Assuntos
Colite , Doenças Inflamatórias Intestinais , Camundongos , Humanos , Animais , Linfócitos T/metabolismo , Apelina/metabolismo , Modelos Animais de Doenças , Colite/patologia , Doenças Inflamatórias Intestinais/metabolismo , Sulfato de Dextrana , Camundongos Endogâmicos C57BL , Linfócitos T CD4-PositivosRESUMO
Crohn's disease is an inflammatory disease of the gut caused by a complex interplay among genetic, microbial, and environmental factors. The intestinal tract is constantly exposed to metals and other trace elements ingested as food. Synchrotron radiation-induced X-ray fluorescence spectroscopy and X-ray absorption fine structure analysis revealed the deposition of nickel particles within Crohn's disease tissue specimens. After nickel particle stimulation, THP-1 cells showed filopodia formation and autophagic vacuoles containing lipid bodies. Nickel particles precipitated colitis in mice bearing mutations of the IBD susceptibility protein A20/TNFAIP3. Nickel particles also exacerbated dextran sulfate sodium-induced colitis in mice harboring myeloid cell-specific Atg5 deficiency. These findings illustrate that nickel particle ingestion may worsen Crohn's disease by perturbing autophagic processes in the intestine, providing new insights into environmental factors in Crohn's disease pathogenesis.
Assuntos
Doença de Crohn/patologia , Progressão da Doença , Inflamação/patologia , Intestinos/patologia , Níquel/toxicidade , Animais , Autofagia/efeitos dos fármacos , Proteína 5 Relacionada à Autofagia/metabolismo , Sulfato de Dextrana , Suscetibilidade a Doenças , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Macrófagos/ultraestrutura , Camundongos Endogâmicos C57BL , Células THP-1 , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismoRESUMO
Expression of mucin MUC2, a component of the colonic mucus layer, plays a crucial role in intestinal homeostasis. Here, we describe a new regulator of MUC2 expression, the deubiquitinase ZRANB1 (Trabid). A ZRANB1 mutation changing cysteine to serine in amino acid position 443, affects ubiquitination. To analyze ZRANB1 function in the intestine, we generated Zranb1 C443S mutant knock-in (Zranb1C443S/C443S) mice using the CRISPR/Cas9 system. Zranb1C443S/C443S mice exhibited decreased mRNA expression and MUC2 production. Colonic organoids from Zranb1C443S/C443S mice displayed decreased Muc2 mRNA expression following differentiation into goblet cells. Finally, we analyzed dextran sulfate sodium-induced colitis to understand ZRANB1's role in intestinal inflammation. Zranb1C443S/C443S mice with colitis exhibited significant weight loss, reduced colon length, and worsening clinical and pathological scores, indicating that ZRANB1 contributes to intestinal homeostasis. Together, these results suggest that ZRANB1 regulates MUC2 expression and intestinal inflammation, which may help elucidating the pathogenesis of inflammatory bowel disease and developing new therapeutics targeting ZRANB1.
Assuntos
Colite , Mucosa Intestinal , Proteases Específicas de Ubiquitina , Animais , Colite/induzido quimicamente , Colite/genética , Colite/metabolismo , Cisteína/metabolismo , Enzimas Desubiquitinantes/metabolismo , Sulfato de Dextrana/toxicidade , Inflamação/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Mucinas/metabolismo , Muco/metabolismo , RNA Mensageiro/genética , Serina/metabolismo , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismoRESUMO
Nickel, the most frequent contact allergy cause, is widely used for various metallic materials and medical devices. Autophagy is an intracellular protein degradation system and contributes to metal recycling. However, it is unclear the functions of nickel in autophagy. We here demonstrated that NiCl2 induced microtubule-associated protein 1 light chain 3 (LC3)-II and LC3 puncta, markers of autophagosomes. Bafilomycin A1 (BafA1) treatment did not enhance LC3 puncta under NiCl2 stimulation, suggesting that NiCl2 did not induce autophagic flux. In addition, NiCl2 promotes the accumulation of SQSTM1/p62 and increased SQSTM1/p62 colocalization with lysosomal-associated membrane protein 1 (LAMP1). These data indicated that NiCl2 attenuates autophagic flux. Interestingly, NiCl2 induced the expression of the high-molecular-weight (MW) form of SQSTM1/p62. Inhibition of NiCl2-induced reactive oxygen species (ROS) reduced the high-MW SQSTM1/p62. We also showed that NiCl2-induced ROS activate transglutaminase (TG) activity. We found that transglutaminase 2 (TG2) inhibition reduced high-MW SQSTM1/p62 and SQSTM1/p62 puncta under NiCl2 stimulation, indicating that TG2 regulates SQSTM1/p62 protein homeostasis under NiCl2 stimulation. Our study demonstrated that nickel ion regulates autophagy flux and TG2 restricted nickel-dependent proteostasis.
RESUMO
Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) expressed in T cells may regulate immune responses in the gut. In addition to T cells, B cells are also an important population in the gut-associated lymphoid tissues that orchestrate mucosal homeostasis. However, the role of CEACAM1 in B cells has not been elucidated. We herein analyzed mature B cells to determine the functions of CEACAM1. Flow cytometry revealed high expression of CEACAM1 on B cells in secondary lymphoid tissues. Cytokine production induced by activation of B cell receptor (BCR) signaling was suppressed by CEACAM1 signaling in contrast to that associated with either Toll-like receptor 4 or CD40 signaling. Confocal microscopy revealed co-localization of CEACAM1 and BCR when activated with anti-Igµ F(ab')2 fragment. Overexpression of CEACAM1 in a murine B cell line, A20, resulted in reduced expressions of activation surface markers with decreased Ca2+ influx after BCR signal activation. Overexpression of CEACAM1 suppressed BCR signal cascade in A20 cells in association with decreased spontaneous proliferation. Our results suggest that CEACAM1 can regulate BCR-mediated mature B cell activation in lymphoid tissues. Therefore, further studies of this molecule may lead to greater insights into the mechanisms of immune responses within peripheral tissues and the potential treatment of inflammatory diseases.
Assuntos
Antígenos CD/metabolismo , Moléculas de Adesão Celular/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Animais , Linfócitos B/metabolismo , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Citocinas/biossíntese , Feminino , Camundongos Endogâmicos C57BLRESUMO
Intraepithelial lymphocytes (IELs) are very unique in the intestinal immune system. They include γδT cells and CD4-CD8-TCRαß+T cells (double negative: DNT), both of which are specific for the intestine, in addition to CD4+ and CD8+ T cells. IELs exist within the monolayer of the intestinal epithelial cells and dynamically move between lamina propria (LP) and intraepithelial (IE) region. The localization and movement patterns of IEL subsets and the regulatory factors have been unknown. Here, we developed a novel in vitro live imaging system and quantified the motility and morphological changes among subsets of IELs. We identified CD8αα as the key regulatory factor. IELs, especially γδ and DNT cells, showed amoeboid shape and frequent morphological change, while most T cells in MLN or SP showed round shape in vitro. TCR signal, IL-15, gut microbes, CCL25, and integrin αEß7 expression were non-essential for IEL movement in vitro. CD8αα+ cells showed higher motility and larger morphological changes than CD8αα- cells. Adoptive transferred CD8αα+CD4-IELs localized to IE region of recipient NSG mice, while CD8αα-CD4-IELs localized to the LP. Our results showed that the CD8αα/TL signal is essential for the localization of IELs to IE region in vivo. CD8αα/TL may be an effective target to increase the number of IELs, which protects against intestinal infection, allergy, tumorigenesis or inflammation.
Assuntos
Antígenos CD8/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos Intraepiteliais/citologia , Linfócitos Intraepiteliais/imunologia , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos/classificação , Movimento Celular/imunologia , Forma Celular , Quimiocinas CC/metabolismo , Feminino , Imunidade nas Mucosas , Interleucina-15/metabolismo , Intestino Delgado/citologia , Intestino Delgado/imunologia , Linfócitos Intraepiteliais/classificação , Microscopia Intravital , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Camundongos TransgênicosRESUMO
In Japan and other Asian countries, increased fat uptake induced by a westernized diet is thought to be associated with an increased incidence of inflammatory bowel disease, colorectal cancer and food allergies; however, the mechanism for this remains unclear. High-fat diet (HFD)-fed mice are common animal models used to examine the effect of fat intake in vivo. HFDs are reported to exacerbate DSS-induced colitis and intestinal tumorigenesis, but the effect of HFDs on the intestines before disease induction is often overlooked. We found that the intestinal and gut-associated lymphoid tissue (GALT) morphology of HFD-fed mice differed from that of standard diet (SD)-fed mice. To clarify the mechanism by which fat intake increases intestinal diseases, we analyzed the morphological and immunological aspects of the intestines of HFD-fed mice as well as the molecular mechanisms and physiology. Feeding an HFD for 3 weeks induced atrophy of the small intestine, colon and GALT and reduced the number of small intestinal intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs). Feeding an HFD for only one day reduced the number of small intestinal (SI)-IELs and SI-LPLs. The effect of feeding a 3-week HFD continued for 2 weeks after returning to the SD. The effect of the HFD on the intestinal immune system was independent of the gut microbes. We hypothesized that the cytotoxicity of the abundant HFD-derived free fatty acids in the intestinal lumen impairs the intestinal immune system. Both saturated and unsaturated free fatty acids were toxic to intestinal T-cells in vitro. Orally administering free fatty acids reduced the number of SI-IELs and LPLs. Using a lipase inhibitor to reduce the luminal free fatty acids attenuated the HFD-induced changes in the intestinal immune system, while using a statin to reduce the serum free fatty acids did not. Thus, HFD-induced free fatty acids damaged the intestines; this effect was termed "intestinal lipotoxicity". Because sustained reduction of SI-LPLs after HFD feeding exacerbated indomethacin-induced small intestinal damage, lipotoxicity to the human intestines incurred by consuming a westernized diet in Japan may increase intestinal diseases such as IBD, colorectal cancer or food allergies.
Assuntos
Dieta Hiperlipídica , Ácidos Graxos não Esterificados/toxicidade , Sistema Imunitário/patologia , Mucosa Intestinal/patologia , Animais , Atrofia , Colo/patologia , Ácidos Graxos não Esterificados/sangue , Comportamento Alimentar , Microbioma Gastrointestinal/efeitos dos fármacos , Sistema Imunitário/efeitos dos fármacos , Indometacina , Mucosa Intestinal/efeitos dos fármacos , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/patologia , Contagem de Linfócitos , Linfócitos/efeitos dos fármacos , Tecido Linfoide/efeitos dos fármacos , Tecido Linfoide/patologia , Masculino , Camundongos Endogâmicos C57BLRESUMO
Although previous studies have suggested that appendix seems to be involved in the colitis, the role of this in the pathogenesis remains unclear. In this study, we assessed the importance of appendiceal lymphoid follicles, specifically the cecal patches (CP) in mice, using an experimental colitis model. Treatment with oxazolone resulted in ulcerations particularly at CP with follicular expansion as well as colitis. The colitis was attenuated by either appendectomy or the absence of mature B cells. We therefore established an intravital imaging system accompanied by the fluorescence resonance energy transfer technology to analyze the dynamic immune response of CP B cells. Our observation revealed frequent Ca2+ signaling in CP B cells during the early phase of colitis development. These findings suggested that the CP B cells may be involved in the pathogenesis of colitis including inflammatory bowel diseases in humans.
Assuntos
Apêndice/imunologia , Ceco/imunologia , Colite/imunologia , Colo/imunologia , Estruturas Linfoides Terciárias/imunologia , Animais , Apêndice/diagnóstico por imagem , Apêndice/patologia , Linfócitos B/imunologia , Linfócitos B/patologia , Sinalização do Cálcio , Ceco/diagnóstico por imagem , Ceco/patologia , Colite/induzido quimicamente , Colite/diagnóstico por imagem , Colite/patologia , Colo/diagnóstico por imagem , Colo/patologia , Modelos Animais de Doenças , Humanos , Microscopia Intravital , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Oxazolona , Estruturas Linfoides Terciárias/diagnóstico por imagem , Estruturas Linfoides Terciárias/patologiaRESUMO
Genome-wide association studies have identified autophagy-related susceptibility genes for inflammatory bowel disease (IBD); however, whether autophagy regulators can be utilized as therapeutic targets remains unclear. To identify novel microtubule-associated protein 1 light chain 3 (LC3)-interacting proteins in intestinal epithelial cells (IECs), we isolated primary IECs from green fluorescent protein (GFP)-LC3 mice. We performed immunoprecipitation with a GFP antibody and then analyzed co-immunoprecipitates by mass spectrometry. HADHA was identified as an LC3-interacting protein from primary IECs. The HADHA gene encodes the alpha subunit of the mitochondrial trifunctional protein. Given that HADHA catalyzes the last three steps of mitochondrial beta-oxidation of long-chain fatty acids, we investigated whether long-chain fatty acids induce autophagy in IECs. We found that palmitic acid induced autophagy in DLD-1, HT29, and HCT116 cells. HADHA was expressed in not only the mitochondria but also the cytosol. LC3 puncta co-localized with HADHA, which were enhanced by palmitic acid stimulation. However, LC3 puncta did not co-localize with Tom20, suggesting that HADHA was induced to associate with LC3 puncta at sites other than the mitochondria. Thus, HADHA may have extra-mitochondrial functions. Furthermore, we found that palmitic acid induced cell death in IECs, which was accelerated by bafilomycin A and chloroquine. These findings suggested that palmitic acid-induced autophagy supports the survival of IECs. Taken together, these results suggested that HADHA is involved in long-chain fatty acid-induced autophagy in IECs, thus providing new insights into the pathology of IBD and revealing novel therapeutic targets of IBD.
Assuntos
Autofagia/fisiologia , Ácidos Graxos/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteína Mitocondrial Trifuncional/metabolismo , Animais , Alcaloides de Berberina/farmacologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Subunidades ProteicasRESUMO
BACKGROUND: Spinal surgery is classified as a moderate risk for DVT. The occurrence of DVT after various spinal surgical procedures was reviewed retrospectively, and the perioperative risk factors in the high-risk group were identified. In addition, the administration of the factor Xa inhibitor to DVT subjects with unstable thrombosis was evaluated to reveal its effectiveness in the prevention of PTE and postoperative complications. METHODS: This study included 588 subjects who underwent lumbar spine surgery. The patient population consisted of the following four groups: the fracture group (F group), the laminectomy group (La group), the TLIF group (T group), and the long fusion group (Lo group). Bilateral lower limb venous ultrasonography was performed on the day before surgery, the day after surgery, and one week after surgery. The incidence of DVT was determined for each group and potential risk factors were evaluated in the group with the highest incidence of DVT. Subjects with DVT who had unstable thrombosis received anticoagulant therapy (factor Xa inhibitor) and their treatment results were assessed. RESULTS: The overall incidence of DVT was 32.3% (190/588). A significantly high incidence of DVT was observed in the Lo group (54.3%; 75/138). Logistic regression and ROC analysis of potential risk factors in the Lo group identified a D-dimer value of 19.5 ug/ml at one week postoperatively as a risk factor of DVT (p = 0.02; odds ratio, 4.09; 95% CI, 2.82-7.88). Overall, 15.8% of subjects (30/190) received anticoagulant therapy. These subjects experienced neither PTE nor epidural hematoma. A follow-up ultrasonography performed at three weeks postoperatively detected the disappearance/resolution of DVT in 86.7% of these subjects (26/30). CONCLUSION: The incidence of DVT varied according to the invasiveness of the procedure. Successful management of DVT hinges on preoperative risk management involving prophylactic treatment and early diagnosis, in order to avoid PTE and other complications.
Assuntos
Vértebras Lombares/cirurgia , Procedimentos Ortopédicos/efeitos adversos , Doenças da Coluna Vertebral/cirurgia , Tromboembolia Venosa/tratamento farmacológico , Tromboembolia Venosa/epidemiologia , Adulto , Idoso , Análise de Variância , Anticoagulantes/administração & dosagem , Estudos de Coortes , Feminino , Seguimentos , Humanos , Incidência , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Procedimentos Ortopédicos/métodos , Complicações Pós-Operatórias/diagnóstico por imagem , Complicações Pós-Operatórias/tratamento farmacológico , Estudos Retrospectivos , Medição de Risco , Doenças da Coluna Vertebral/diagnóstico por imagem , Doenças da Coluna Vertebral/patologia , Fusão Vertebral , Tromboembolia Venosa/diagnósticoRESUMO
RIPK3 is a key molecule for necroptosis, initially characterized by necrotic cell death morphology and the activation of autophagy. Cell death and autophagic signaling are believed to tightly regulate each other. However, the associated recruitment of signaling proteins remains poorly understood. p62/sequestosome-1 is a selective autophagy substrate and a selective receptor for ubiquitinated proteins. In this study, we illustrated that both mouse and human RIPK3 mediate p62 cleavage and that RIPK3 interacts with p62, resulting in complex formation. In addition, RIPK3-dependent p62 cleavage is restricted by the inhibition of caspases, especially caspase-8. Moreover, overexpression of A20, a ubiquitin-editing enzyme and an inhibitor of caspase-8 activity, inhibits RIPK3-dependent p62 cleavage. To further investigate the potential role of RIPK3 in selective autophagy, we analyzed p62-LC3 complex formation, revealing that RIPK3 prevents the localization of LC3 and ubiquitinated proteins to the p62 complex. In addition, RIPK3-dependent p62-LC3 complex disruption is regulated by caspase inhibition. Taken together, these results demonstrated that RIPK3 interacts with p62 and regulates p62-LC3 complex formation. These findings suggested that RIPK3 serves as a negative regulator of selective autophagy and provides new insights into the mechanism by which RIPK3 regulates autophagic signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia , Caspase 8/metabolismo , Regulação Enzimológica da Expressão Gênica , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Apoptose , Inibidores Enzimáticos/química , Células HEK293 , Proteínas de Choque Térmico/metabolismo , Humanos , Camundongos , Fagossomos/metabolismo , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Proteína Sequestossoma-1RESUMO
Although intestinal microbiota are essential for the development of T cell-mediated colitis, it remains undetermined whether they enhance or suppress the chronic extraintestinal inflammation that often complicates inflammatory bowel diseases. In this study, we investigate the role of intestinal microbiota in the development of colitis and extraintestinal manifestations in a mouse model in which colitis was induced in SCID mice by adoptive transfer of CD4(+)CD45RB(high) T cells. Under specific pathogen-free conditions, these mice developed both colitis and extraintestinal interstitial pneumonia, whereas mice given a mixture of antibiotics did not develop colitis, but, surprisingly, developed Th1/Th17-mediated IP. Irrespective of antibiotic treatment, cotransfer of CD4(+)CD25(+) regulatory T cells suppressed the development of pneumonitis and colitis, with all local CD4(+)CD45RB(high) T cell-derived cells converted to CD44(high)CD62L(-)IL-7Rα(high) effector-memory T cells. Retransfer of CD4(+) effector-memory T cells from the lungs of antibiotic-treated mice with IP not only induced IP in both antibiotic-treated and -untreated recipients but also induced colitis in the untreated recipients. In summary, we have established a unique model of Th1/Th17-mediated IP in microbiota-free and antibiotic-treated mice. This model may be valuable in investigating the immunological mechanisms underlying extraintestinal disorders in patients with inflammatory bowel disease.
Assuntos
Colite/imunologia , Modelos Animais de Doenças , Doenças Pulmonares Intersticiais/imunologia , Células Th1/imunologia , Células Th17/imunologia , Transferência Adotiva , Animais , Colite/complicações , Colite/microbiologia , Citocinas/análise , Citocinas/biossíntese , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imuno-Histoquímica , Doenças Pulmonares Intersticiais/complicações , Doenças Pulmonares Intersticiais/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos SCIDRESUMO
Notch signaling plays an essential role in the proliferation and differentiation of intestinal epithelial cells (IECs). We have previously shown that Notch signaling is up-regulated in the inflamed mucosa of ulcerative colitis (UC) and thereby plays an indispensable role in tissue regeneration. Here we show that in addition to Notch signaling, STAT3 signaling is highly activated in the inflamed mucosa of UC. Forced expression of the Notch target gene Hes1 dramatically enhanced the IL-22-mediated STAT3-dependent transcription in human IECs. This enhancement of STAT3-dependent transcription was achieved by the extended phosphorylation of STAT3 by Hes1. Microarray analysis revealed that Hes1-mediated enhancement of IL-22-STAT3 signaling significantly increased the induction of genes encoding antimicrobial peptides, such as REG1A, REG3A and REG3G, in human IECs. Conversely, the reduction of Hes1 protein levels with a γ-secretase inhibitor significantly down-regulated the induction of those genes in IECs, resulting in a markedly poor response to IL-22. Our present findings identify a new role for the molecular function of Hes1 in which the protein can interact with cytokine signals and regulate the immune response of IECs.
Assuntos
Anti-Infecciosos/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Enterócitos/metabolismo , Proteínas de Homeodomínio/metabolismo , Interleucinas/farmacologia , Fator de Transcrição STAT3/metabolismo , Transcrição Gênica/efeitos dos fármacos , Antígenos de Neoplasias/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular , Colite Ulcerativa/genética , Colite Ulcerativa/patologia , Enterócitos/efeitos dos fármacos , Enterócitos/patologia , Proteínas de Homeodomínio/genética , Humanos , Inflamação/patologia , Proteínas Associadas a Pancreatite , Fosforilação/efeitos dos fármacos , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Transcrição HES-1 , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Interleucina 22RESUMO
BACKGROUND AND AIMS: The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour. METHODS: Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. RESULTS: We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. CONCLUSIONS: The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus.
Assuntos
Células Epiteliais/citologia , Fluorescência , Intestinos/citologia , Organoides/citologia , Células-Tronco/citologia , Células Cultivadas , Células HEK293 , HumanosRESUMO
OBJECTIVE: Interleukin (IL)-7 is mainly produced in bone marrow (BM) that forms the niche for B cells. We previously demonstrated that BM also retains pathogenic memory CD4 T cells in murine models of inflammatory bowel disease (IBD). However, it remains unknown whether BM-derived IL-7 is sufficient for the development of IBD and which cells form the niche for colitogenic memory CD4 T cells in BM. DESIGN: To address these questions, we developed mice in which IL-7 expression was specific for BM, and identified colitis-associated IL-7-expressing mesenchymal stem cells (MSC) in the BM. RESULTS: IL-7-/-×RAG-1-/- mice injected with BM cells from IL-7+/+×RAG-1-/- mice, but not from IL-7-/-×RAG-1-/- mice, expressed IL-7 in BM, but not in their colon, and developed colitis when injected with CD4+CD45RBhigh T cells. Cultured BM MSC stably expressed a higher level of IL-7 than that of primary BM cells. IL-7-sufficient, but not IL-7-deficient, BM MSC supported upregulation of Bcl-2 in, and homeostatic proliferation of, colitogenic memory CD4 T cells in vitro. Notably, IL-7-/-×RAG-1-/- mice transplanted with IL-7-sufficient, but not IL-7-deficient, BM MSC expressed IL-7 in BM, but not in their colon, and developed colitis when transplanted with CD4+CD45RBhigh T cells. CONCLUSIONS: We demonstrate for the first time that BM MSC are a major source of IL-7 and play a pathological role in IBD by forming the niche for colitogenic CD4 memory T cells in BM.
Assuntos
Células da Medula Óssea/imunologia , Linfócitos T CD4-Positivos/imunologia , Colite Ulcerativa/imunologia , Interleucina-7/biossíntese , Células-Tronco Mesenquimais/imunologia , Transferência Adotiva , Animais , Transplante de Medula Óssea , Células Cultivadas , Técnicas de Cocultura , Colite Ulcerativa/patologia , Colo/imunologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/imunologia , Memória Imunológica/imunologia , Interleucina-7/genética , Ativação Linfocitária/imunologia , Transplante de Células-Tronco Mesenquimais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genéticaRESUMO
Recent advances in research suggest that aging has a controllable chronic inflammatory disease aspect. Aging systemic T cells, which secrete pro-inflammatory factors, affect surrounding somatic cells, and accelerate the aging process through chronic inflammation, have attracted attention as potential therapeutic targets in aging. On the other hand, there are few reports on the aging of the intestinal immune system, which differs from the systemic immune system in many ways. In the current study, we investigated the age-related changes in the intestinal immune system, particularly in T cells. The most significant changes were observed in the CD4+ T cells in the small intestinal IEL, with a marked increase in this fraction in old mice and reduced expression of CD27 and CD28, which are characteristic of aging systemic T cells. The proliferative capacity of aging IEL CD4+ T cells was significantly more reduced than that of aging systemic T cells. Transcriptome analysis showed that the expression of inflammatory cytokines was not upregulated, whereas Cd8α, NK receptors, and Granzymes were upregulated in aging IEL CD4+ T cells. Functional analysis showed that aging IEL T cells had a higher cytotoxic function against intestinal tumor organoids in vitro than young IEL T cells. scRNAseq revealed that splenic T cells show a transition from naïve to memory T cells, whereas intestinal T cells show the emergence of a CD8αα+CD4+ T cell fraction in aged mice, which is rarely seen in young cells. Further analysis of the aging IEL CD4+ T cells showed that two unique subsets are increased that are distinct from the systemic CD4+ T cells. Subset 1 has a pro-inflammatory component, with expression of IFNγ and upregulation of NFkB signaling pathways. Subset 2 does not express IFNγ, but upregulates inhibitory molecules and nIEL markers. Expression of granzymes and Cd8a was common to both. These fractions were in opposite positions in the clustering by UMAP and had different TCR repertoires. They may be involved in the suppression of intestinal aging and longevity through anti-tumor immunity, elimination of senescent cells and stressed cells in the aging environment. This finding could be a breakthrough in aging research.
Assuntos
Linfócitos Intraepiteliais , Camundongos , Animais , Linfócitos T CD4-Positivos , Granzimas , Subpopulações de Linfócitos T , Análise de Célula ÚnicaRESUMO
BACKGROUND & AIMS: T helper (Th) 17 cells that express the retinoid-related orphan receptor (ROR) γt contribute to the development of colitis in mice, yet are found in normal and inflamed intestine. We investigated their development and functions in intestines of mice. METHODS: We analyzed intestinal Th17 cells in healthy and inflamed intestinal tissues of mice. We analyzed expression of lymphotoxin (LT)α by Th17 cells and lymphoid tissue inducer-like cells. RESULTS: LTα(-/-) and RORγt(-/-) mice had significantly lower percentages of naturally occurring Th17 cells in the small intestine than wild-type mice. Numbers of CD3(-)CD4(+/-)interleukin-7Rα(+)c-kit(+)CCR6(+)NKp46(-) lymphoid tissue inducer-like cells that produce interleukin-17A were increased in LTα(-/-) and LTα(-/-) × recombination activating gene (RAG)-2(-/-) mice, compared with wild-type mice, but were absent from RORγt(-/-) mice. Parabiosis of wild-type and LTα(-/-) mice and bone marrow transplant experiments revealed that LTα-dependent gut-associated lymphoid tissue structures are required for generation of naturally occurring Th17 cells. However, when wild-type or LTα(-/-) CD4(+)CD45RB(high) T cells were transferred to RAG-2(-/-) or LTα(-/-)×RAG-2(-/-) mice, all groups, irrespective of the presence or absence of LTα on the donor or recipient cells, developed colitis and generated Th1, Th17, and Th17/Th1 cells. RAG-2(-/-) mice that received a second round of transplantation, with colitogenic but not naturally occurring Th17 cells, developed intestinal inflammation. The presence of naturally occurring Th17 cells in the colons of mice inhibited development of colitis after transfer of CD4(+)CD45RB(high) T cells and increased the numbers of Foxp3(+) cells derived from CD4(+)CD45RB(high) T cells. CONCLUSIONS: Gut-associated lymphoid tissue structures are required to generate naturally occurring Th17 cells that have regulatory activities in normal intestines of mice, but not for colitogenic Th17 and Th17/Th1 cells during inflammation.
Assuntos
Colite/imunologia , Mucosa Intestinal/imunologia , Tecido Linfoide/imunologia , Linfotoxina-alfa/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Células Th17/metabolismo , Animais , Complexo CD3/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/transplante , Colo/imunologia , Proteínas de Ligação a DNA/genética , Fatores de Transcrição Forkhead/metabolismo , Interferon gama/metabolismo , Interleucina-17/metabolismo , Interleucinas/metabolismo , Intestino Delgado/imunologia , Células Matadoras Naturais/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Contagem de Linfócitos , Tecido Linfoide/metabolismo , Linfotoxina-alfa/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Parabiose , Células Th1 , Células Th17/imunologia , Interleucina 22RESUMO
We have previously demonstrated that IL-7 is essential for the persistence of colitis as a survival factor of colitogenic IL-7Rα-expressing memory CD4(+) T cells. Because IL-7Rα is broadly expressed on various immune cells, it is possible that the persistence of colitogenic CD4(+) T cells is affected by other IL-7Rα-expressing non-T cells. To test this hypothesis, we conducted two adoptive transfer colitis experiments using IL-7Rα(-/-) CD4(+)CD25(-) donor cells and IL-7Rα(-/-) × RAG-2(-/-) recipient mice, respectively. First, IL-7Rα expression on colitic lamina propria (LP) CD4(+) T cells was significantly higher than on normal LP CD4(+) T cells, whereas expression on other colitic LP immune cells, (e.g., NK cells, macrophages, myeloid dendritic cells) was conversely lower than that of paired LP cells in normal mice, resulting in predominantly higher expression of IL-7Rα on colitogenic LP CD4(+) cells, which allows them to exclusively use IL-7. Furthermore, RAG-2(-/-) mice transferred with IL-7Rα(-/-) CD4(+)CD25(-) T cells did not develop colitis, although LP CD4(+) T cells from mice transferred with IL-7Rα(-/-) CD4(+)CD25(-) T cells were differentiated to CD4(+)CD44(high)CD62L(-) effector-memory T cells. Finally, IL-7Rα(-/-) × RAG-2(-/-) mice transferred with CD4(+)CD25(-) T cells developed colitis similar to RAG-2(-/-) mice transferred with CD4(+)CD25(-) T cells. These results suggest that IL-7Rα expression on colitogenic CD4(+) T cells, but not on other cells, is essential for the development of chronic colitis. Therefore, therapeutic approaches targeting the IL-7/IL-7R signaling pathway in colitogenic CD4(+) T cells may be feasible for the treatment of inflammatory bowel diseases.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Colite/imunologia , Memória Imunológica , Receptores de Interleucina-7/biossíntese , Regulação para Cima/imunologia , Transferência Adotiva , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/transplante , Células Cultivadas , Doença Crônica , Colite/genética , Colite/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Feminino , Predisposição Genética para Doença , Memória Imunológica/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interleucina-7/deficiência , Receptores de Interleucina-7/genética , Regulação para Cima/genéticaRESUMO
BACKGROUND: The organoids therapy for ulcerative colitis (UC) is under development. It is important to dissect how the engrafted epithelium can provide benefits for overcoming the vulnerability to inflammation. We mainly focused on the deliverability of sulfomucin, which is reported to play an important role in epithelial function. METHODS: We analyzed each segment of colon epithelium to determine differences in sulfomucin production in both mice and human. Subsequently, we transplanted organoids established from sulfomucin-enriched region into the injured recipient epithelium following dextran sulfate sodium-induced colitis and analyzed the engrafted epithelium in mouse model. RESULTS: In human normal colon, sulfomucin production was increased in proximal colon, whereas it was decreased in the inflammatory region of UC. In murine colon epithelium, increased sulfomucin production was found in cecum compared to distal small intestine and proximal colon. RNA sequencing analysis revealed that several key genes associated with sulfomucin production such as Papss2 and Slc26a1 were enriched in isolated murine cecum crypts. Then we established murine cecum organoids and transplanted them into the injured epithelium of distal colon. Although the expression of sulfomucin was temporally decreased in cecum organoids, its secretion was restored again in the engrafted patches after transplantation. Finally, we verified a part of mechanisms controlling sulfomucin production in human samples. CONCLUSION: This study illustrated the deliverability of sulfomucin in the disease-relevant grafting model to design sulfomucin-producing epithelial units in severely injured distal colon. The current study is the basis for the better promotion of organoids transplantation therapy for refractory UC.