RESUMO
Metabolic dysfunction-associated steatohepatitis (MASH) is the most prevalent cause of liver disease worldwide, with a single approved therapeutic. Previous research has shown that interleukin-22 (IL-22) can suppress ß-cell stress, reduce local islet inflammation, restore appropriate insulin production, reverse hyperglycemia, and ameliorate insulin resistance in preclinical models of diabetes. In clinical trials long-acting forms of IL-22 have led to increased proliferation in the skin and intestine, where the IL-22RA1 receptor is highly expressed. To maximise beneficial effects whilst reducing the risk of epithelial proliferation and cancer, we designed short-acting IL-22-bispecific biologic drugs that successfully targeted the liver and pancreas. Here we show 10-fold lower doses of these bispecific biologics exceed the beneficial effects of native IL-22 in multiple preclinical models of MASH, without off-target effects. Treatment restores glycemic control, markedly reduces hepatic steatosis, inflammation, and fibrogenesis. These short-acting IL-22-bispecific targeted biologics are a promising new therapeutic approach for MASH.
Assuntos
Fígado Gorduroso , Interleucina 22 , Interleucinas , Fígado , Pâncreas , Interleucinas/metabolismo , Animais , Fígado/metabolismo , Fígado/patologia , Fígado/efeitos dos fármacos , Pâncreas/patologia , Pâncreas/metabolismo , Pâncreas/efeitos dos fármacos , Humanos , Camundongos , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Resistência à Insulina , Receptores de Interleucina/metabolismoRESUMO
The IL-22RA1 receptor is highly expressed in the pancreas, and exogenous IL-22 has been shown to reduce endoplasmic reticulum and oxidative stress in human pancreatic islets and promote secretion of high-quality insulin from beta-cells. However, the endogenous role of IL-22RA1 signaling on these cells remains unclear. Here, we show that antibody neutralisation of IL-22RA1 in cultured human islets leads to impaired insulin quality and increased cellular stress. Through the generation of mice lacking IL-22ra1 specifically on pancreatic alpha- or beta-cells, we demonstrate that ablation of murine beta-cell IL-22ra1 leads to similar decreases in insulin secretion, quality and islet regeneration, whilst increasing islet cellular stress, inflammation and MHC II expression. These changes in insulin secretion led to impaired glucose tolerance, a finding more pronounced in female animals compared to males. Our findings attribute a regulatory role for endogenous pancreatic beta-cell IL-22ra1 in insulin secretion, islet regeneration, inflammation/cellular stress and appropriate systemic metabolic regulation.
Assuntos
Glucose , Homeostase , Células Secretoras de Insulina , Insulina , Camundongos Knockout , Receptores de Interleucina , Animais , Células Secretoras de Insulina/metabolismo , Receptores de Interleucina/metabolismo , Receptores de Interleucina/genética , Feminino , Humanos , Masculino , Insulina/metabolismo , Camundongos , Glucose/metabolismo , Secreção de Insulina , Camundongos Endogâmicos C57BL , Interleucina 22 , Intolerância à Glucose/metabolismo , Interleucinas/metabolismo , Interleucinas/genética , Envelhecimento/metabolismoRESUMO
Bacteria adapt to selective pressure in their immediate environment in multiple ways. One mechanism involves the acquisition of independent mutations that disable or modify a key pathway, providing a signature of adaptation via convergent evolution. Extra-intestinal pathogenic Escherichia coli (ExPEC) belonging to sequence type 95 (ST95) represent a global clone frequently associated with severe human infections including acute pyelonephritis, sepsis, and neonatal meningitis. Here, we analysed a publicly available dataset of 613 ST95 genomes and identified a series of loss-of-function mutations that disrupt cellulose production or its modification in 55.3% of strains. We show the inability to produce cellulose significantly enhances ST95 invasive infection in a rat model of neonatal meningitis, leading to the disruption of intestinal barrier integrity in newborn pups and enhanced dissemination to the liver, spleen and brain. Consistent with these observations, disruption of cellulose production in ST95 augmented innate immune signalling and tissue neutrophil infiltration in a mouse model of urinary tract infection. Mutations that disrupt cellulose production were also identified in other virulent ExPEC STs, Shigella and Salmonella, suggesting a correlative association with many Enterobacteriaceae that cause severe human infection. Together, our findings provide an explanation for the emergence of hypervirulent Enterobacteriaceae clones.
Assuntos
Infecções por Escherichia coli , Proteínas de Escherichia coli , Meningite , Camundongos , Animais , Ratos , Humanos , Virulência/genética , Infecções por Escherichia coli/microbiologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fatores de Virulência/genética , FilogeniaRESUMO
Major histocompatibility complex (MHC) II is dynamically expressed on mucosal epithelial cells and is induced in response to inflammation and parasitic infections, upon exposure to microbiota, and is increased in chronic inflammatory diseases. However, the regulation of epithelial cell-specific MHC II during homeostasis is yet to be explored. We discovered a novel role for IL-22 in suppressing epithelial cell MHC II partially via the regulation of endoplasmic reticulum (ER) stress, using animals lacking the interleukin-22-receptor (IL-22RA1), primary human and murine intestinal and respiratory organoids, and murine models of respiratory virus infection or with intestinal epithelial cell defects. IL-22 directly downregulated interferon-γ-induced MHC II on primary epithelial cells by modulating the expression of MHC II antigen A α (H2-Aα) and Class II transactivator (Ciita), a master regulator of MHC II gene expression. IL-22RA1-knockouts have significantly higher MHC II expression on mucosal epithelial cells. Thus, while IL-22-based therapeutics improve pathology in chronic disease, their use may increase susceptibility to viral infections.
Assuntos
Interleucinas , Complexo Principal de Histocompatibilidade , Humanos , Animais , Camundongos , Estresse do Retículo Endoplasmático , Células Epiteliais , Interleucina 22RESUMO
BACKGROUND & AIMS: MUC13 cell surface mucin is highly expressed on the mucosal surface throughout the intestine, yet its role against bacterial infection is unknown. We investigated how MUC13 impacts Salmonella typhimurium (S Tm) infection and elucidated its mechanisms of action. METHODS: Muc13-/- and wild-type littermate mice were gavaged with 2 isogenic strains of S Tm after pre-conditioning with streptomycin. We assessed clinical parameters, cecal histology, local and systemic bacterial load, and proinflammatory cytokines after infection. Cecal enteroids and epithelial cell lines were used to evaluate the mechanism of MUC13 activity after infection. The interaction between bacterial SiiE and MUC13 was assessed by using siiE-deficient Salmonella. RESULTS: S Tm-infected Muc13-/- mice had increased disease activity, histologic damage, and higher local and systemic bacterial loads. Mechanistically, we found that S Tm binds to MUC13 through its giant SiiE adhesin and that MUC13 acts as a pathogen-binding decoy shed from the epithelial cell surface after pathogen engagement, limiting bacterial invasion. In addition, MUC13 reduces epithelial cell death and intestinal barrier breakdown by enhancing nuclear factor kappa B signaling during infection, independent of its decoy function. CONCLUSIONS: We show for the first time that MUC13 plays a critical role in antimicrobial defense against pathogenic S Tm at the intestinal mucosal surface by both acting as a releasable decoy limiting bacterial invasion and reducing pathogen-induced cell death. This further implicates the cell surface mucin family in mucosal defense from bacterial infection.
Assuntos
Infecções Bacterianas , Mucinas , Animais , Camundongos , Infecções Bacterianas/genética , Infecções Bacterianas/metabolismo , Células Epiteliais/metabolismo , Mucosa Intestinal/patologia , Mucinas/metabolismo , Salmonella typhimurium/metabolismoRESUMO
Nanoparticle based permeation enhancers have the potential to improve the oral delivery of biologics. Recently, solid silica nanoparticles were discovered to improve the intestinal permeability of peptides and proteins via transient opening of the gut epithelium. In this study, we have developed small-sized (â¼60 nm) virus-like silica nanoparticles (VSNP) as a reversible and next generation non-toxic permeation enhancer for oral delivery of biologics. Our results show that the anionic VSNP showed a better permeation-enhancing effect than the same sized spherical Stöber silica nanoparticles (â¼60 nm) by enhancing the apparent insulin permeability by 1.3-fold in the Caco-2 monolayer model and by 1.2-fold in the Caco-2/MTX-HT-29 co-culture model. In vivo experiments in healthy mice demonstrated that anionic VSNP significantly enhanced the permeation of fluorescently labelled 4 kDa dextran after oral administration compared to Stöber nanoparticles and positively charged VSNP. The results indicated that the nanoscale surface roughness is an important consideration when designing nanoparticle-based permeation enhancers. Overall, our study shows for the first time that small-sized (â¼60 nm) VSNP with nanoscale surface roughness can be used as a non-toxic permeation enhancer for oral delivery of therapeutic peptides and proteins.
Assuntos
Nanopartículas , Dióxido de Silício , Humanos , Camundongos , Animais , Células CACO-2 , Dióxido de Silício/metabolismo , Mucosa Intestinal/metabolismo , Peptídeos/química , Administração Oral , Nanopartículas/químicaRESUMO
BACKGROUND & AIMS: MUC1 is abnormally expressed in colorectal cancer, including colitis-associated colorectal cancer (CAC), but its role in tumorigenesis is unclear. This study investigated MUC1's effects in murine models of colitis and CAC and elucidated mechanisms of action. METHODS: Colitis and CAC were induced in mice by exposure to dextran sodium sulfate or azoxymethane plus dextran sodium sulphate. Clinical parameters, immune cell infiltration, and tumor development were monitored throughout disease progression. Experiments in knockout mice and bone marrow chimeras were combined with an exploration of immune cell abundance and function. RESULTS: Deficiency of Muc1 suppressed inflammation, inhibited tumor progression, increased abundance of CD8+ T lymphocytes, and reduced abundance of macrophages in colon tumors. Bone marrow chimeras showed promotion of CAC was primarily mediated by Muc1-expressing hematopoietic cells, and that MUC1 promoted a pro-tumoral immunosuppressive macrophage phenotype within tumors. Mechanistic studies revealed that Muc1 deficiency remarkably reduced interleukin-6 levels in the colonic tissues and tumors that was mainly produced by infiltrating macrophages at day 21, 42, and 85. In bone marrow-derived macrophages, MUC1 promoted responsiveness to chemoattractant and promoted activation into a phenotype with high Il6 and Ido1 expression, secreting factors which inhibited CD8+ T cell proliferation. MUC1 potently drives macrophages to produce interleukin-6, which in turn drives a pro-tumorigenic activation of signal transducer and activator of transcription 3 in colon epithelial tumor and stromal cells, ultimately increasing the occurrence and development of CAC. CONCLUSIONS: Our findings provide cellular and molecular mechanisms for the pro-tumorigenic functions of MUC1 in the inflamed colon. Therapeutic strategies to inhibit MUC1 signal transduction warrant consideration for the prevention or therapy of CAC.
Assuntos
Neoplasias Associadas a Colite , Interleucina-6 , Ativação de Macrófagos , Mucina-1 , Fator de Transcrição STAT3 , Animais , Azoximetano/toxicidade , Carcinogênese , Fatores Quimiotáticos , Colite/induzido quimicamente , Colite/genética , Colite/imunologia , Neoplasias Associadas a Colite/genética , Neoplasias Associadas a Colite/imunologia , Neoplasias do Colo/genética , Neoplasias do Colo/imunologia , Sulfato de Dextrana/toxicidade , Interleucina-6/genética , Interleucina-6/imunologia , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Camundongos , Camundongos Knockout , Mucina-1/genética , Mucina-1/imunologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologiaRESUMO
The mucosal surfaces that form the boundary between the external environment and the underlying tissue are protected by a mucus barrier. Mucin glycoproteins, both secreted and cell surface mucins, are the major components of the barrier. They can exclude pathogens and toxins while hosting the commensal bacteria. In this review, we highlight the dynamic function of the mucins and mucus during infection, how this mucosal barrier is regulated, and how pathogens have evolved mechanisms to evade this defence system.
Assuntos
Mucinas , Muco , Bactérias/metabolismo , Glicoproteínas/metabolismo , Mucinas/metabolismo , Mucosa/microbiologia , Muco/metabolismoRESUMO
The cytokine Interleukin (IL)-20 belongs to the IL-10 superfamily. IL-20 levels are reported to increase in the intestines of Ulcerative Colitis (UC) patients, however not much is known about its effects on intestinal epithelial cells. Here, we investigated the influence of IL-20 on intestinal epithelial cell lines and primary intestinal organoid cultures. By using chemical-induced (dextran sodium sulphate; DSS) colitis and a spontaneous model of colitis (Winnie mice), we assess whether recombinant IL-20 treatment is beneficial in reducing/improving pathology. Following stimulation with IL-20, intestinal primary organoids from wild-type and Winnie mice increased the expression of ERK1/2. However, this was lost when cells were differentiated into secretory goblet cells. Importantly, IL-20 treatment significantly reduced endoplasmic reticulum (ER) stress, as measured by spliced-XBP1 in epithelial cells, and this effect was lost in the goblet cells. IL-20 treatment in vivo in the DSS and Winnie models had minimal effects on pathology, but a decrease in macrophage activation was noted. Taken together, these data suggest a possible, but subtle role of IL-20 on epithelial cells in vivo. The therapeutic potential of IL-20 could be harnessed by the development of a targeted therapy or combination therapy to improve the healing of the mucosal barrier.
Assuntos
Colite Ulcerativa , Interleucinas , Animais , Camundongos , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/genética , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Interleucinas/farmacologia , Mucosa Intestinal/metabolismo , Intestinos/patologia , Sistema de Sinalização das MAP Quinases , Camundongos Endogâmicos C57BLRESUMO
Rationale: The alarmins IL-33 and HMGB1 (high mobility group box 1) contribute to type 2 inflammation and asthma pathogenesis. Objectives: To determine whether P2Y13-R (P2Y13 receptor), a purinergic GPCR (G protein-coupled receptor) and risk allele for asthma, regulates the release of IL-33 and HMGB1. Methods: Bronchial biopsy specimens were obtained from healthy subjects and subjects with asthma. Primary human airway epithelial cells (AECs), primary mouse AECs, or C57Bl/6 mice were inoculated with various aeroallergens or respiratory viruses, and the nuclear-to-cytoplasmic translocation and release of alarmins was measured by using immunohistochemistry and an ELISA. The role of P2Y13-R in AEC function and in the onset, progression, and exacerbation of experimental asthma was assessed by using pharmacological antagonists and mice with P2Y13-R gene deletion. Measurements and Main Results: Aeroallergen exposure induced the extracellular release of ADP and ATP, nucleotides that activate P2Y13-R. ATP, ADP, and aeroallergen (house dust mite, cockroach, or Alternaria antigen) or virus exposure induced the nuclear-to-cytoplasmic translocation and subsequent release of IL-33 and HMGB1, and this response was ablated by genetic deletion or pharmacological antagonism of P2Y13. In mice, prophylactic or therapeutic P2Y13-R blockade attenuated asthma onset and, critically, ablated the severity of a rhinovirus-associated exacerbation in a high-fidelity experimental model of chronic asthma. Moreover, P2Y13-R antagonism derepressed antiviral immunity, increasing IFN-λ production and decreasing viral copies in the lung. Conclusions: We identify P2Y13-R as a novel gatekeeper of the nuclear alarmins IL-33 and HMGB1 and demonstrate that the targeting of this GPCR via genetic deletion or treatment with a small-molecule antagonist protects against the onset and exacerbations of experimental asthma.
Assuntos
Asma/imunologia , Proteína HMGB1/metabolismo , Interleucina-33/metabolismo , Receptores Purinérgicos P2/metabolismo , Animais , Asma/metabolismo , Asma/fisiopatologia , Biomarcadores/metabolismo , Estudos de Casos e Controles , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Macromolecular therapeutics of biological origin, also known as biologics, have become one of the fastest-growing classes of drugs for management of a range of chronic and acute conditions. The majority of approved biologics are administered via the parenteral route and are thus expensive, have low patient compliance, and have high systemic toxicity. Therefore, tremendous efforts have been devoted to the development of carriers for oral delivery of biologics. This review evaluates key chemical (e.g. pH and enzymes) and physiological challenges to oral biologics delivery. We review the conventional formulation strategies and their limitations, followed by a detailed account of the progress on the use of nanocarriers used for oral biologics delivery, covering organic and inorganic nanocarriers. Lastly, we discuss limitations and opportunities presented by these emerging nanomaterials in oral biologics delivery.
Assuntos
Produtos Biológicos , Nanopartículas , Administração Oral , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Humanos , Nanopartículas/químicaRESUMO
Type 2 diabetes (T2D) is a well-known risk factor for tuberculosis (TB), but little is known about pre-diabetes and the relative contribution of impaired glucose tolerance vs. obesity towards susceptibility to TB. Here, we developed a preclinical model of pre-diabetes and TB. Mice fed a high fat diet (HFD) for 12 weeks presented with impaired glucose tolerance and hyperinsulinemia compared to mice fed normal chow diet (NCD). Infection with M. tuberculosis (Mtb) H37Rv after the onset of dysglycemia was associated with significantly increased lung pathology, lower concentrations of TNF-α, IFN-γ, IFN-ß and IL-10 and a trend towards higher bacterial burden at 3 weeks post infection. To determine whether the increased susceptibility of pre-diabetic mice to TB is reversible and is associated with dysglycemia or increased body fat mass, we performed a diet reversal experiment. Pre-diabetic mice were fed a NCD for 10 additional weeks (HFD/NCD) at which point glucose tolerance was restored, but body fat mass remained higher compared to control mice that consumed NCD throughout the entire experiment (NCD/NCD). Upon Mtb infection HFD/NCD mice had significantly lower bacterial burden compared to NCD/NCD mice and this was accompanied by restored IFN-γ responses. Our findings demonstrate that pre-diabetes increases susceptibility to TB, but a high body mass index without dysglycemia is protective. This murine model offers the opportunity to further study the underlying immunological, metabolic and endocrine mechanisms of this association.
Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Intolerância à Glucose , Estado Pré-Diabético , Tuberculose , Tecido Adiposo , Animais , Dieta Hiperlipídica/efeitos adversos , Camundongos , Camundongos Endogâmicos C57BL , Índice de Gravidade de DoençaRESUMO
Respiratory syncytial virus (RSV) bronchiolitis is a leading cause of infant hospitalization and mortality. We previously identified that prostaglandin D2 (PGD2), released following RSV infection of primary human airway epithelial cells or pneumonia virus of mice (PVM) infection of neonatal mice, elicits pro- or antiviral innate immune responses as a consequence of D-type prostanoid receptor 2 (DP2) or DP1 activation, respectively. Here, we sought to determine whether treatment with the DP1 agonist BW245c decreases the severity of bronchiolitis in PVM-infected neonatal mice. Consistent with previous findings, BW245c treatment increased IFN-λ production and decreased viral load in week 1 of the infection. However, unexpectedly, BW245c treatment increased mortality in week 2 of the infection. This increased morbidity was associated with viral spread to the parenchyma, an increased cellular infiltrate of TNF-α-producing cells (neutrophils, monocytes, and CD4+ T cells), and the heightened production of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1ß. These phenotypes, as well as the increased mortality, were significantly attenuated following the administration of anti-TNF-α to PVM-infected, BW245c-treated mice. In summary, pharmacological activation of the DP1 receptor in PVM-infected neonatal mice boosts antiviral innate and adaptive immunity, however, this is ultimately detrimental, as a consequence of increased TNF-α-induced morbidity and mortality.
Assuntos
Suscetibilidade a Doenças , Receptores de Prostaglandina/agonistas , Infecções Respiratórias/etiologia , Infecções Respiratórias/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Viroses/etiologia , Viroses/metabolismo , Doença Aguda , Animais , Biomarcadores , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças/imunologia , Camundongos , Monócitos/imunologia , Monócitos/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/mortalidade , Índice de Gravidade de Doença , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Viroses/diagnósticoRESUMO
Intestinal epithelial cell endoplasmic reticulum (ER) stress has been implicated in intestinal inflammation. It remains unclear whether ER stress is an initiator of or a response to inflammation. Winnie mice, carrying a Muc2 gene mutation resulting in intestinal goblet cell ER stress, develop spontaneous colitis with a depleted mucus barrier and increased bacterial translocation. This study aims to determine whether the microbiota was required for the development of Winnie colitis, and whether protein misfolding itself can initiate inflammation directly in absence of the microbiota. To assess the role of microbiota in driving Winnie colitis, WT and Winnie mice on the same background were rederived into the germ-free facility and housed in the Trexler-type soft-sided isolators. The colitis phenotype of these mice was assessed and compared to WT and Winnie mice housed within a specific pathogen-free facility. We found that Winnie colitis was substantially reduced but not abolished under germ-free conditions. Expression of inflammatory cytokine genes was reduced but several chemokines remained elevated in absence of microbiota. Concomitantly, ER stress was also diminished, although mucin misfolding persisted. RNA-Seq revealed that Winnie differentiated colon organoids have decreased expression of the negative regulators of the inflammatory response compared to WT. This data along with the increase in Mip2a chemokine expression, suggests that the epithelial cells in the Winnie mice are more responsive to stimuli. Moreover, the data demonstrate that intestinal epithelial intrinsic protein misfolding can prime an inflammatory response without initiating the unfolded protein response in the absence of the microbiota. However, the microbiota is necessary for the amplification of colitis in Winnie mice. Genetic predisposition to mucin misfolding in secretory cells initiates mild inflammatory signals. However, the inflammatory signal sets a forward-feeding cycle establishing progressive inflammation in the presence of microbiota.Abbreviations: Endoplasmic Reticulum: ER; Mucin-2: Muc-2; GF: Germ-Free; Inflammatory Bowel Disease: IBD.
Assuntos
Colite Ulcerativa/microbiologia , Microbioma Gastrointestinal , Mucosa Intestinal/fisiopatologia , Animais , Colite Ulcerativa/patologia , Colite Ulcerativa/fisiopatologia , Citocinas/genética , Citocinas/metabolismo , Estresse do Retículo Endoplasmático , Células Epiteliais/metabolismo , Vida Livre de Germes , Células Caliciformes/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Camundongos , Mucina-2/química , Mucina-2/genética , Celulas de Paneth/metabolismo , Dobramento de ProteínaRESUMO
BACKGROUND & AIMS: Chronic bowel inflammation increases the risk of colon cancer; colitis-associated cancer (CAC). Thiopurine treatments are associated with a reduction in dysplasia and CAC in inflammatory bowel disease (IBD). Abnormal Wnt/ß-catenin signalling is characteristic of >90% of colorectal cancers. Immunosuppression by thiopurines is via Rac1 GTPase, which also affects Wnt/ß-catenin signalling. Autophagy is implicated in colonic tumors, and topical delivery of the thiopurine thioguanine (TG) is known to alleviate colitis and augment autophagy. This study investigated the effects of TG in a murine model of CAC and potential mechanisms. METHODS: Colonic dysplasia was induced by exposure to azoxymethane (AOM) and dextran sodium sulfate (DSS) in wild-type (WT) mice and mice harboring intestinal epithelial cell-specific deletion of autophagy related 7 gene (Atg7ΔIEC). TG or vehicle was administered intrarectally, and the effect on tumor burden and ß-catenin activity was assessed. The mechanisms of action of TG were investigated in vitro and in vivo. RESULTS: TG ameliorated DSS colitis in wild-type but not Atg7ΔIEC mice, demonstrating that anti-inflammatory effects of locally delivered TG are autophagy-dependent. However, TG inhibited CAC in both wild-type and Atg7ΔIEC mice. This was associated with decreased ß-catenin activation/nuclear translocation demonstrating that TG's inhibition of tumorigenesis occurred independently of anti-inflammatory and pro-autophagic actions. These results were confirmed in cell lines, and the dependency on Rac1 GTPase was demonstrated by siRNA knockdown and overexpression of constitutively active Rac1. CONCLUSIONS: Our findings provide evidence for a new mechanism that could be exploited to improve CAC chemoprophylactic approaches.
Assuntos
Neoplasias Associadas a Colite/prevenção & controle , Colite/tratamento farmacológico , Tioguanina/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Administração Retal , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Azoximetano/administração & dosagem , Azoximetano/toxicidade , Células CACO-2 , Colite/induzido quimicamente , Colite/imunologia , Colite/patologia , Neoplasias Associadas a Colite/imunologia , Neoplasias Associadas a Colite/patologia , Colo/efeitos dos fármacos , Colo/patologia , Sulfato de Dextrana/administração & dosagem , Sulfato de Dextrana/toxicidade , Técnicas de Silenciamento de Genes , Células HCT116 , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Mercaptopurina/farmacologia , Mercaptopurina/uso terapêutico , Camundongos , Camundongos Transgênicos , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Tioguanina/uso terapêutico , beta Catenina/análise , beta Catenina/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
The cell surface mucin MUC1 is an important host factor limiting Helicobacter pylori (H. pylori) pathogenesis in both humans and mice by providing a protective barrier and modulating mucosal epithelial and leukocyte responses. The aim of this study was to establish the time-course of molecular events in MUC1-modulated gene expression profiles in response to H. pylori infection in wild type (WT) and MUC1-deficient mice using microarray-determined mRNA expression, gene network analysis and Ingenuity Pathway Analysis (IPA). A time-course over the first 72 h of infection showed significantly higher mucosal loads of bacteria at 8 h of infection in Muc1-/- mice compared with WT, confirming its importance in the early stages of infection (P = 0.0003). Microarray analysis revealed 266 differentially expressed genes at one or more time-points over 72 h in the gastric mucosa of Muc1-/- mice compared with WT control using a threshold of 2-fold change. The SPINK1 pancreatic cancer canonical pathway was strongly inhibited in Muc1-/- mice compared with WT at sham and 8 h infection (P = 6.08E-14 and P = 2.25 E-19, respectively) but potently activated at 24 and 72 h post-infection (P = 1.38E-22 and P = 5.87E-13, respectively). The changes in this pathway are reflective of higher expression of genes mediating digestion and absorption of lipids, carbohydrates, and proteins at sham and 8 h infection in the absence of MUC1, but that this transcriptional signature is highly down regulated as infection progresses in the absence of MUC1. Uninfected Muc1-/- gastric tissue was highly enriched for expression of factors involved in lipid metabolism and 8 h infection further activated this network compared with WT. As infection progressed, a network of antimicrobial and anti-inflammatory response genes was more highly activated in Muc1-/- than WT mice. Key target genes identified by time-course microarrays were independently validated using RT-qPCR. These results highlight the dynamic interplay between the host and H. pylori, and the role of MUC1 in host defense, and provide a general picture of changes in cellular gene expression modulated by MUC1 in a time-dependent manner in response to H. pylori infection.
Assuntos
Mucosa Gástrica , Infecções por Helicobacter , Mucina-1/genética , Animais , Helicobacter pylori , Camundongos , TranscriptomaRESUMO
INTRODUCTION: IL-22 may have a role in the alleviation of the metabolic syndrome (MetS) via protection of pancreatic beta and endothelial cells from oxidative and lipid-induced damage. We aimed to investigate the effects of moderate-intensity continuous training (MICT) and different volumes of high-intensity interval training (HIIT) on changes in circulating IL-22. METHODS: This was a sub-study of the "Exercise in the prevention of Metabolic Syndrome" (EX-MET) a multi-center, randomized trial. This study used data collected at the Brisbane site. Thirty-nine individuals with MetS were randomized to one of three 16-wk interventions: 1) MICT (n=10, 30min at 60-70% HR peak, 5x/wk); 2) 4HIIT (n=13, 4x4min at 85-95% HR peak, interspersed with 3min of active recovery at 50-70% HR peak, 3x/wk); or 3) 1HIIT (n=16, 1x4min at 85-95% HR peak, 3x/wk). Serum IL-22 concentration was measured following a 12-hr fast via an enzyme linked immunosorbent assay, before and after the intervention. MetS severity, insulin resistance (IR), visceral adipose tissue (VAT), and cardiorespiratory fitness (CRF) were also measured via MetS z-score, HOMA-IR, dual-energy X-ray absorptiometry, and indirect calorimetry (maximal exercise test), respectively. RESULTS: The median (IQR) IL-22% changes from pre- to post-intervention in the MICT, 4HIIT, and 1HIIT groups were -17% (-43.0% to 31.3%), +16.5% (-18.9% to 154.9%), and +15.9% (-28.7% to 46.1%), respectively. Although there were no significant between-group differences in IL-22 concentration change, there was a medium-to-large group × time interaction effect [F(2,35)=2.08, p=0.14, η2=0.14]. CONCLUSION: Although there was no statistically significant between-group difference in IL-22 change, the study suggests that different exercise intensities may have opposing effects on IL-22 concentration in individuals with MetS.
RESUMO
Diabetes mellitus is a known susceptibility factor for severe influenza virus infections. However, the mechanisms that underlie this susceptibility remain incompletely understood. Here, the effects of high glucose levels on influenza severity were investigated using an in vitro model of the pulmonary epithelial-endothelial barrier as well as an in vivo murine model of type II diabetes. In vitro we show that high glucose conditions prior to IAV infection increased virus-induced barrier damage. This was associated with an increased pro-inflammatory response in endothelial cells and the subsequent damage of the epithelial junctional complex. These results were subsequently validated in vivo. This study provides the first evidence that hyperglycaemia may increase influenza severity by damaging the pulmonary epithelial-endothelial barrier and increasing pulmonary oedema. These data suggest that maintaining long-term glucose control in individuals with diabetes is paramount in reducing the morbidity and mortality associated with influenza virus infections.
Assuntos
Células Endoteliais/fisiologia , Células Epiteliais/fisiologia , Glucose/metabolismo , Vírus da Influenza A/fisiologia , Influenza Humana/virologia , Pulmão/fisiopatologia , Animais , Modelos Animais de Doenças , Células Endoteliais/virologia , Células Epiteliais/virologia , Feminino , Humanos , Pulmão/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
There is an increasing interest in studying the crosstalk between tumor-associated adipose tissue and tumor progression. In proximity to the primary site of kidney tumors, perinephric adipose tissue has direct contact with cancer cells when kidney cancer becomes invasive. To mimic the perinephric adipose tissue microenvironment, we applied the liquid overlay-based technique, which cost-effectively generated functional adipocyte spheroids using mesenchymal stem cells isolated from human perinephric adipose tissue. Thereafter, we co-cultured adipocyte spheroids with unpolarized macrophages and discovered an M2 phenotype skew in macrophages. Moreover, we discovered that, in the presence of adipocyte spheroids, M2 macrophages exhibited stronger invasive capacity than M1 macrophages. We further showed that the perinephric adipose tissue sampled from metastatic kidney cancer exhibited high expression of M2 macrophages. In conclusion, the liquid overlay-based technique can generate a novel three-dimensional platform enabling investigation of the interactions of adipocytes and other types of cells in a tumor microenvironment.
Assuntos
Adipócitos/citologia , Adipogenia , Tecido Adiposo/citologia , Técnicas de Cultura de Células/instrumentação , Células-Tronco Mesenquimais/citologia , Adipócitos/patologia , Tecido Adiposo/patologia , Técnicas de Cultura de Células/economia , Células Cultivadas , Microambiente Celular , Técnicas de Cocultura/economia , Técnicas de Cocultura/instrumentação , Humanos , Neoplasias Renais/patologia , Macrófagos/citologia , Macrófagos/patologia , Células-Tronco Mesenquimais/patologia , Esferoides Celulares/citologia , Esferoides Celulares/patologia , Células Tumorais CultivadasRESUMO
Rationale: Non-cystic fibrosis bronchiectasis is characterized by airway mucus accumulation and sputum production, but the role of mucus concentration in the pathogenesis of these abnormalities has not been characterized.Objectives: This study was designed to: 1) measure mucus concentration and biophysical properties of bronchiectasis mucus; 2) identify the secreted mucins contained in bronchiectasis mucus; 3) relate mucus properties to airway epithelial mucin RNA/protein expression; and 4) explore relationships between mucus hyperconcentration and disease severity.Methods: Sputum samples were collected from subjects with bronchiectasis, with and without chronic erythromycin administration, and healthy control subjects. Sputum percent solid concentrations, total and individual mucin concentrations, osmotic pressures, rheological properties, and inflammatory mediators were measured. Intracellular mucins were measured in endobronchial biopsies by immunohistochemistry and gene expression. MUC5B (mucin 5B) polymorphisms were identified by quantitative PCR. In a replication bronchiectasis cohort, spontaneously expectorated and hypertonic saline-induced sputa were collected, and mucus/mucin concentrations were measured.Measurements and Main Results: Bronchiectasis sputum exhibited increased percent solids, total and individual (MUC5B and MUC5AC) mucin concentrations, osmotic pressure, and elastic and viscous moduli compared with healthy sputum. Within subjects with bronchiectasis, sputum percent solids correlated inversely with FEV1 and positively with bronchiectasis extent, as measured by high-resolution computed tomography, and inflammatory mediators. No difference was detected in MUC5B rs35705950 SNP allele frequency between bronchiectasis and healthy individuals. Hypertonic saline inhalation acutely reduced non-cystic fibrosis bronchiectasis mucus concentration by 5%.Conclusions: Hyperconcentrated airway mucus is characteristic of subjects with bronchiectasis, likely contributes to disease pathophysiology, and may be a target for pharmacotherapy.