RESUMO
Intraepithelial lymphocytes (IELs) are T cells important for the maintenance of barrier integrity in the intestine. Colon IELs are significantly reduced in both MyD88-deficient mice and those lacking an intact microbiota, suggesting that MyD88-mediated detection of bacterial products is important for the recruitment and/or retention of these cells. Here, using conditionally deficient MyD88 mice, we show that myeloid cells are the key mediators of TCRαß+ IEL recruitment to the colon. Upon exposure to luminal bacteria, myeloid cells produce sphingosine-1-phosphate (S1P) in a MyD88-dependent fashion. TCRαß+ IEL recruitment may be blocked using the S1P receptor antagonist FTY720, confirming the importance of S1P in the recruitment of TCRαß+ IELs to the colon epithelium. Finally, using the TNFΔARE/+ model of Crohn's-like bowel inflammation, we show that disruption of colon IEL recruitment through myeloid-specific MyD88 deficiency results in reduced pathology. Our results illustrate one mechanism for recruitment of a subset of IELs to the colon.
Assuntos
Colo , Mucosa Intestinal , Linfócitos Intraepiteliais , Lisofosfolipídeos , Camundongos Knockout , Células Mieloides , Fator 88 de Diferenciação Mieloide , Receptores de Antígenos de Linfócitos T alfa-beta , Esfingosina , Animais , Lisofosfolipídeos/metabolismo , Camundongos , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Colo/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Células Mieloides/imunologia , Células Mieloides/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Linfócitos Intraepiteliais/imunologia , Linfócitos Intraepiteliais/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Camundongos Endogâmicos C57BL , Cloridrato de Fingolimode/farmacologia , Doença de Crohn/imunologiaRESUMO
Hepatic innate immune function plays an important role in the pathogenesis of many diseases. Importantly, a growing body of literature has firmly established the spatial heterogeneity of hepatocyte metabolic function; however, whether innate immune function is zonated remains unknown. To test this question, we exposed adult C57BL/6 mice to endotoxemia, and hepatic tissue was assessed for the acute phase response (APR). The zone-specific APR was evaluated in periportal and pericentral/centrilobular hepatocytes isolated using digitonin perfusion and on hepatic tissue using RNAscope and immunohistochemistry. Western blot, EMSA, chromatin immunoprecipitation, and immunohistochemistry were used to determine the role of the transcription factor NF-κB in mediating hepatic C-reactive protein (CRP) expression. Finally, the ability of mice lacking the NF-κB subunit p50 (p50-/-) to raise a hepatic APR was evaluated. We found that endotoxemia induces a hepatocyte transcriptional APR in both male and female mice, with Crp, Apcs, Fga, Hp, and Lbp expression being enriched in pericentral/centrilobular hepatocytes. Focusing our work on CRP expression, we determined that NF-κB transcription factor subunit p50 binds to consensus sequence elements present in the murine CRP promoter. Furthermore, pericentral/centrilobular hepatocyte p50 nuclear translocation is temporally associated with zone-specific APR during endotoxemia. Lastly, the APR and CRP expression is blunted in endotoxemic p50-/- mice. These results demonstrate that the murine hepatocyte innate immune response to endotoxemia includes zone-specific activation of transcription factors and target gene expression. These results support further study of zone-specific hepatocyte innate immunity and its role in the development of various disease states.
Assuntos
Endotoxemia , NF-kappa B , Masculino , Feminino , Animais , Camundongos , NF-kappa B/metabolismo , Proteína C-Reativa/metabolismo , Camundongos Endogâmicos C57BL , Fígado/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Imunidade InataRESUMO
Background: Combination therapy with lenvatinib plus programmed death-1 (PD-1) immune checkpoint blockades (ICBs) is under investigation in many solid tumors, including thyroid cancer. Lenvatinib is known to reduce angiogenesis and may overturn the immunosuppressive effects of vascular endothelial growth factor in the tumor microenvironment. Previous studies investigating the effects of VEGF receptor inhibition on the immune response were performed in rapidly growing tumor models where immune equilibrium is not established before treatment. We hypothesize that physiologically relevant preclinical models are necessary to define mechanisms of resistance to immune-targeted combination therapies. Methods: We utilized the TPO-CreER/BrafV600E/wt/Trp53Δex2-10/Δex2-10 inducible transgenic model of advanced thyroid cancer to investigate lenvatinib treatment in the context of an anti-PD-1 ICB. Following tumor establishment, 3.5 months postinduction, mice were treated with high- (10 mg/kg) or low-dose (2 mg/kg) lenvatinib, anti-PD-1, or combination of lenvatinib with anti-PD-1. Tumor volume and lung metastases were assessed in each group. Immune infiltrate was characterized by flow cytometry and immunohistochemistry, and TCRß sequencing was performed to further investigate the T cell response. Results: Both low- and high-dose lenvatinib reduced tumor volume, while anti-PD-1 had no effect, alone or in combination. Although both low- and high-dose lenvatinib reduced vascular density, low-dose lenvatinib was superior in controlling tumor size. Lung metastases and survival were not improved with therapy despite the effects of lenvatinib on primary tumor size. Low-dose lenvatinib treatment led to a subtle reduction in the dominant Ly6G+CD11b+ myeloid cell population and was associated with increased CD4+ T cell infiltrate and enrichment in 4-1BB+ and granzyme B+ CD4+ T cells and FoxP3+ regulatory T cells. Polyclonal T cell expansion was evident in the majority of mice, suggesting that a tumor-specific T cell response was generated. Conclusions: The effects of lenvatinib on the immune response were most pronounced in mice treated with low-dose lenvatinib, suggesting that dose should be considered in clinical application. While the immune-modulating potential of lenvatinib is encouraging, alterations in the immune milieu and T cell activation status were insufficient to sustain durable tumor regression, even with added anti-PD-1. Additional studies are necessary to develop more effective combination approaches in low-mutation burden tumors, such as thyroid cancer.
Assuntos
Resistência a Medicamentos , Inibidores de Checkpoint Imunológico , Compostos de Fenilureia/administração & dosagem , Quinolinas/administração & dosagem , Neoplasias da Glândula Tireoide/patologia , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , Animais , Antineoplásicos/uso terapêutico , Quimioterapia Combinada , Humanos , Camundongos , Modelos Animais , Neoplasias da Glândula Tireoide/induzido quimicamenteRESUMO
BACKGROUND AND AIMS: Chronically administered parenteral nutrition (PN) in patients with intestinal failure carries the risk for developing PN-associated cholestasis (PNAC). We have demonstrated that farnesoid X receptor (FXR) and liver X receptor (LXR), proinflammatory interleukin-1 beta (IL-1ß), and infused phytosterols are important in murine PNAC pathogenesis. In this study we examined the role of nuclear receptor liver receptor homolog 1 (LRH-1) and phytosterols in PNAC. APPROACH AND RESULTS: In a C57BL/6 PNAC mouse model (dextran sulfate sodium [DSS] pretreatment followed by 14 days of PN; DSS-PN), hepatic nuclear receptor subfamily 5, group A, member 2/LRH-1 mRNA, LRH-1 protein expression, and binding of LRH-1 at the Abcg5/8 and Cyp7a1 promoter was reduced. Interleukin-1 receptor-deficient mice (Il-1r-/- /DSS-PN) were protected from PNAC and had significantly increased hepatic mRNA and protein expression of LRH-1. NF-κB activation and binding to the LRH-1 promoter were increased in DSS-PN PNAC mice and normalized in Il-1r-/- /DSS-PN mice. Knockdown of NF-κB in IL-1ß-exposed HepG2 cells increased expression of LRH-1 and ABCG5. Treatment of HepG2 cells and primary mouse hepatocytes with an LRH-1 inverse agonist, ML179, significantly reduced mRNA expression of FXR targets ATP binding cassette subfamily C member 2/multidrug resistance associated protein 2 (ABCC2/MRP2), nuclear receptor subfamily 0, groupB, member 2/small heterodimer partner (NR0B2/SHP), and ATP binding cassette subfamily B member 11/bile salt export pump (ABCB11/BSEP). Co-incubation with phytosterols further reduced expression of these genes. Similar results were obtained by suppressing the LRH-1 targets ABCG5/8 by treatment with small interfering RNA, IL-1ß, or LXR antagonist GSK2033. Liquid chromatography-mass spectrometry and chromatin immunoprecipitation experiments in HepG2 cells showed that ATP binding cassette subfamily G member 5/8 (ABCG5/8) suppression by GSK2033 increased the accumulation of phytosterols and reduced binding of FXR to the SHP promoter. Finally, treatment with LRH-1 agonist, dilauroyl phosphatidylcholine (DLPC) protected DSS-PN mice from PNAC. CONCLUSIONS: This study suggests that NF-κB regulation of LRH-1 and downstream genes may affect phytosterol-mediated antagonism of FXR signaling in the pathogenesis of PNAC. LRH-1 could be a potential therapeutic target for PNAC.
Assuntos
Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Colestase/etiologia , Lipoproteínas/metabolismo , NF-kappa B/metabolismo , Nutrição Parenteral/efeitos adversos , Fitosteróis/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Colestase/metabolismo , Imunoprecipitação da Cromatina , Modelos Animais de Doenças , Cromatografia Gasosa-Espectrometria de Massas , Técnicas de Silenciamento de Genes , Células Hep G2 , Humanos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Pelvic organ prolapse is common, but the underlying etiologies are poorly understood, which limits our current prevention and treatment options. OBJECTIVE: Our primary objective was to compare the uterosacral ligament histologic features in women with and without prolapse using the novel pelvic organ prolapse histologic quantification system. Our secondary aim was to determine whether composite histologic findings in uterosacral ligaments are associated with prolapse risk factors. STUDY DESIGN: This was a prospective cohort study in which paracervical uterosacral ligament biopsies were performed at the time of hysterectomy for primary prolapse or other benign gynecologic indications and processed for histologic evaluation. The pelvic organ prolapse quantification system was used to determine the prolapse stage. In this study, 9 prominent histologic features were semiquantitatively scored using the pelvic organ prolapse histologic quantification system in a blinded fashion and compared between prolapse and control groups. Unbiased principal component analysis of these scores was independently performed to identify potential relationships between histologic measures and prolapse risk factors. RESULTS: The histologic scores of 81 prolapse and 33 control ligaments were analyzed. Compared with the control group, women in the prolapse group were significantly older and more likely to be in the menopausal phase. There was no difference in the number of vaginal deliveries, body mass index, hormone use, or smoking status between the groups. To control for baseline differences, patients were also stratified by age over 40 years and menopausal status. Compared with the control group, the prolapse ligaments in the premenopausal group had significantly more loss of smooth muscle fibers within the fascicles (P<.001), increased inflammatory infiltrates of neutrophils within the tissue and perineural inflammatory cells (P<.01 and P=.04, respectively), and reduced neointimal hyperplasia (P=.02). Prolapse ligaments in the postmenopausal group exhibited elevated adipose content compared with that of the control group (P=.05). Amount of fibrillar collagen, total nonvascular smooth muscle, and muscle fiber vesicles of prolapse ligaments did not differ in either the premenopausal or postmenopausal group compared with that of the control group. Unbiased principal component analysis of the histologic scores separated the prolapse ligaments into 3 phenotypes: (1) increased adipose accumulation, (2) increased inflammation, and (3) abnormal vasculature, with variable overlap with controls. Posthoc analysis of these subgroups demonstrated a positive correlation between increasing number of vaginal deliveries and body mass index with increasing adipose content in the adipocyte accumulation and inflammatory phenotype and increasing neointimal hyperplasia in the vascular phenotype. However, only the relationship between vaginal delivery and adipocytes was significant in the adipose phenotype (R2=0.13; P=.04). CONCLUSION: Histologic phenotypes exist in pelvic support ligaments that can be distinguished using the pelvic organ prolapse histologic quantification system and principle component analysis. Vaginal delivery is associated with aberrant adipose accumulation in uterosacral ligaments. Our findings support a multifactorial etiology for pelvic organ prolapse contributing to altered smooth muscle, vasculature, and connective tissue content in crucial pelvic support structures. To confirm these associations and evaluate the biomechanical properties of histologic phenotypes of prolapse, larger studies are warranted. Closing this gap in knowledge will help optimize personalized medicine and help identify targets for prevention and treatment of this complex condition.
Assuntos
Ligamentos/fisiopatologia , Prolapso de Órgão Pélvico/fisiopatologia , Sacro , Útero , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Humanos , Pessoa de Meia-Idade , Fenótipo , Estudos Prospectivos , Fatores de Risco , Índice de Gravidade de DoençaRESUMO
A crucial component of nonalcoholic fatty liver disease (NAFLD) pathogenesis is lipid stress, which may contribute to hepatic inflammation and activation of innate immunity in the liver. However, little is known regarding how dietary lipids, including fat and cholesterol, may facilitate innate immune activation in vivo. We hypothesized that dietary fat and cholesterol drive NAFLD progression to steatohepatitis and hepatic fibrosis by altering the transcription and phenotype of hepatic macrophages. This hypothesis was tested by using RNA-sequencing methods to characterize and analyze sort-purified hepatic macrophage populations that were isolated from mice fed diets with varying amounts of fat and cholesterol. The addition of cholesterol to a high-fat diet triggered hepatic pathology reminiscent of advanced nonalcoholic steatohepatitis (NASH) in humans characterized by signs of cholesterol dysregulation, generation of oxidized low-density lipoprotein, increased recruitment of hepatic macrophages, and significant fibrosis. RNA-sequencing analyses of hepatic macrophages in this model revealed that dietary cholesterol induced a tissue repair and regeneration phenotype in Kupffer cells (KCs) and recruited infiltrating macrophages to a greater degree than fat. Furthermore, comparison of diseased KCs and infiltrating macrophages revealed that these two macrophage subsets are transcriptionally diverse. Finally, direct stimulation of murine and human macrophages with oxidized low-density lipoprotein recapitulated some of the transcriptional changes observed in the RNA-sequencing study. These findings indicate that fat and cholesterol synergize to alter macrophage phenotype, and they also challenge the dogma that KCs are purely proinflammatory in NASH. Conclusion: This comprehensive view of macrophage populations in NASH indicates mechanisms by which cholesterol contributes to NASH progression and identifies potential therapeutic targets for this common disease.
Assuntos
Colesterol na Dieta/efeitos adversos , Células de Kupffer/metabolismo , Fígado/imunologia , Hepatopatia Gordurosa não Alcoólica/etiologia , Animais , Progressão da Doença , Hepatite/etiologia , Células de Kupffer/ultraestrutura , Metabolismo dos Lipídeos , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , TranscriptomaRESUMO
Type 2 diabetes is associated with impaired exercise capacity. Alterations in both muscle perfusion and mitochondrial function can contribute to exercise impairment. We hypothesized that impaired muscle mitochondrial function in type 2 diabetes is mediated, in part, by decreased tissue oxygen delivery and would improve with oxygen supplementation. Ex vivo muscle mitochondrial content and respiration assessed from biopsy samples demonstrated expected differences in obese individuals with (n = 18) and without (n = 17) diabetes. Similarly, in vivo mitochondrial oxidative phosphorylation capacity measured in the gastrocnemius muscle via 31P-MRS indicated an impairment in the rate of ADP depletion with rest (27 ± 6 s [diabetes], 21 ± 7 s [control subjects]; P = 0.008) and oxidative phosphorylation (P = 0.046) in type 2 diabetes after isometric calf exercise compared with control subjects. Importantly, the in vivo impairment in oxidative capacity resolved with oxygen supplementation in adults with diabetes (ADP depletion rate 5.0 s faster, P = 0.012; oxidative phosphorylation 0.046 ± 0.079 mmol/L/s faster, P = 0.027). Multiple in vivo mitochondrial measures related to HbA1c These data suggest that oxygen availability is rate limiting for in vivo mitochondrial oxidative exercise recovery measured with 31P-MRS in individuals with uncomplicated diabetes. Targeting muscle oxygenation could improve exercise function in type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Mitocôndrias Musculares/efeitos dos fármacos , Obesidade/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Oxigênio/administração & dosagem , Adulto , Idoso , Respiração Celular/efeitos dos fármacos , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/terapia , Exercício Físico/fisiologia , Terapia por Exercício/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Obesidade/complicações , Obesidade/terapia , Oxigênio/farmacologia , Consumo de Oxigênio/fisiologia , Comportamento SedentárioRESUMO
Biliary atresia (BA) is a neonatal T cell-mediated, inflammatory, sclerosing cholangiopathy. In the rhesus rotavirus (RRV)-induced neonatal mouse model of BA (murine BA), mice lacking B cells do not develop BA, and the lack of B cells is associated with loss of T-cell and macrophage activation. The aim of this study was to determine the mechanism of B cell-mediated immune activation (antigen presentation versus cytokine production) in murine BA. Normal neonatal B cells in the liver are predominantly at pro-B and pre-B cellular development. However, BA mice exhibit a significant increase in the number and activation status of mature liver B cells. Adoptively transferred B cells into RRV-infected, B cell-deficient mice were able to reinstate T-cell and macrophage infiltration and biliary injury. Nonetheless, neonatal liver B cells were incompetent at antigen presentation to T cells. Moreover, 3-83 immunoglobulin transgenic mice, in which B cells only present an irrelevant antigen, developed BA, indicating a B-cell antigen-independent mechanism. B cells from BA mice produced a variety of innate and adaptive immune cytokines associated with immune activation. In vitro trans-well studies revealed that BA B cells secreted cytokines that activated T cells based on increased expression of T-cell activation marker cluster of differentiation 69. Conclusion: Neonatal liver B cells are highly activated in murine BA and contribute to immune activation through production of numerous cytokines involved in innate and adaptive immunity; this work provides increased knowledge on the capacity of neonatal B cells to contribute to an inflammatory disease through cytokine-mediated mechanisms, and future studies should focus on targeting B cells as a therapeutic intervention in human BA.
Assuntos
Linfócitos B/metabolismo , Ductos Biliares/patologia , Atresia Biliar/imunologia , Citocinas/metabolismo , Imunidade Adaptativa/imunologia , Adolescente , Animais , Animais Recém-Nascidos , Linfócitos B/imunologia , Ductos Biliares/imunologia , Técnicas de Cultura de Células , Criança , Pré-Escolar , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Fígado/imunologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Análise de Sequência de RNA , Baço/imunologiaRESUMO
OBJECTIVES: The aim of the current study was to examine expression and the role, if any, of aldehyde dehydrogenase (ALDH)1B1 in pancreatic adenocarcinoma. METHODS: A tissue microarray of 61 pancreatic cancer patients were evaluated for protein expression of ALDH1B1 by immunohistochemistry. The ALDH1B1 small interfering (RNA) was used to assess the contribution of ALDH1B1 on proliferation of pancreatic cancer cells. RESULTS: In normal human pancreas, ALDH1B1 is abundantly expressed in glandular cells, but sparsely in the ducts (ALDH1B1 immunopositivity = 16.7 ± 1.7). In pancreatic ductal carcinoma, we found high ALDH1B1 expression in ductal cancerous tissues (ALDH1B1 immunopositivity = 197.2 ± 29.4). Analysis of ALDH1B1 expression in a human pancreatic adenocarcinoma tissue microarray showed the greatest expression in tumors that were more invasive. A variation in ALDH1B1 expression was also observed in 16 human pancreatic cancer cell lines. Knockdown of ALDH1B1 caused a 35% reduction in cell growth in the high ALDH1B1-expressing cell lines. CONCLUSIONS: Our data show for the first time that ALDH1B1 is expressed at very high levels in human pancreatic cancer, and it contributes to proliferation in these tumor cells. These data suggest a potential modulatory role for ALDH1B1 in pancreatic cancer.
Assuntos
Aldeído Desidrogenase/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/enzimologia , Neoplasias Pancreáticas/enzimologia , Aldeído Desidrogenase/genética , Família Aldeído Desidrogenase 1 , Aldeído-Desidrogenase Mitocondrial , Animais , Biomarcadores Tumorais/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Camundongos Nus , Invasividade Neoplásica , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Interferência de RNA , Transdução de Sinais , Análise Serial de Tecidos , Transfecção , Carga Tumoral , Regulação para CimaRESUMO
UNLABELLED: Fructose intake from added sugars has been implicated as a cause of nonalcoholic fatty liver disease. Here we tested the hypothesis that fructose may interact with a high-fat diet to induce fatty liver, and to determine if this was dependent on a key enzyme in fructose metabolism, fructokinase. Wild-type or fructokinase knockout mice were fed a low-fat (11%), high-fat (36%), or high-fat (36%) and high-sucrose (30%) diet for 15 weeks. Both wild-type and fructokinase knockout mice developed obesity with mild hepatic steatosis and no evidence of hepatic inflammation on a high-fat diet compared to a low-fat diet. In contrast, wild-type mice fed a high-fat and high-sucrose diet developed more severe hepatic steatosis with low-grade inflammation and fibrosis, as noted by increased CD68, tumor necrosis factor alpha, monocyte chemoattractant protein-1, alpha-smooth muscle actin, and collagen I and TIMP1 expression. These changes were prevented in the fructokinase knockout mice. CONCLUSION: An additive effect of high-fat and high-sucrose diet on the development of hepatic steatosis exists. Further, the combination of sucrose with high-fat diet may induce steatohepatitis. The protection in fructokinase knockout mice suggests a key role for fructose (from sucrose) in this development of steatohepatitis. These studies emphasize the important role of fructose in the development of fatty liver and nonalcoholic steatohepatitis.