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1.
Physiol Genomics ; 56(3): 247-264, 2024 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-38073491

RESUMEN

Chronic intestinal inflammation is a poorly understood manifestation of cystic fibrosis (CF), which may be refractory to ion channel CF transmembrane conductance regulator (CFTR) modulator therapy. People with CF exhibit intestinal dysbiosis, which has the potential for stimulating intestinal and systemic inflammation. CFTR is expressed in organ epithelia, leukocytes, and other tissues. Here, we investigate the contribution of intestinal epithelium-specific loss of Cftr [iCftr knockout (KO)] to dysbiosis and inflammation in mice treated with either of two antiobstructive dietary regimens necessary to maintain CF mouse models [polyethylene glycol (PEG) laxative or a liquid diet (LiqD)]. Feces collected from iCftr KO mice and their wild-type (WT) sex-matched littermates were used to measure fecal calprotectin to evaluate inflammation and to perform 16S rRNA sequencing to characterize the gut microbiome. Fecal calprotectin was elevated in iCftr KO relative to WT mice that consumed either PEG or LiqD. PEG iCftr KO mice did not show a change in α diversity versus WT mice but demonstrated a significant difference in microbial composition (ß diversity) with included increases in the phylum Proteobacteria, the family Peptostreptococcaceae, four genera of Clostridia including C. innocuum, and the mucolytic genus Akkermansia. Fecal microbiome analysis of LiqD-fed iCftr KO mice showed both decreased α diversity and differences in microbial composition with increases in the Proteobacteria family Enterobacteriaceae, Firmicutes families Clostridiaceae and Peptostreptococcaceae, and enrichment of Clostridium perfringens, C. innocuum, C. difficile, mucolytic Ruminococcus gnavus, and reduction of Akkermansia. It was concluded that epithelium-specific loss of Cftr is a major driver of CF intestinal dysbiosis and inflammation with significant similarities to previous studies of pan Cftr KO mice.NEW & NOTEWORTHY Chronic intestinal inflammation is a manifestation of cystic fibrosis (CF), a disease caused by loss of the anion channel CF transmembrane conductance regulator (CFTR) that is expressed in many tissues. This study shows that intestinal epithelial cell-specific loss of CFTR [inducible Cftr knockout (KO)] in mice is sufficient to induce intestinal dysbiosis and inflammation. Experiments were performed on mice consuming two dietary regimens routinely used to prevent obstruction in CF mice.


Asunto(s)
Clostridioides difficile , Fibrosis Quística , Obstrucción Intestinal , Animales , Humanos , Ratones , Clostridioides difficile/genética , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Disbiosis/microbiología , Expectorantes/uso terapéutico , Heces , Inflamación , Complejo de Antígeno L1 de Leucocito/uso terapéutico , Ratones Endogámicos CFTR , Ratones Noqueados , ARN Ribosómico 16S
2.
bioRxiv ; 2023 Jul 26.
Artículo en Inglés | MEDLINE | ID: mdl-37546931

RESUMEN

Chronic intestinal inflammation is a poorly understood manifestation of Cystic Fibrosis (CF), which may be refractory to ion channel CFTR modulator therapy. People with CF exhibit intestinal dysbiosis which has potential for stimulating intestinal and systemic inflammation. CFTR is expressed in organ epithelia and in the leukocyte population. Here, we investigate the contribution of intestinal epithelial-specific loss of Cftr (iCftr KO) to dysbiosis and inflammation in mice treated with either of two anti-obstructive dietary regimens necessary to maintain CF mouse models (PEG laxative or a liquid diet, LiqD). Feces collected from iCftr KO mice and their wildtype (WT) sex-matched littermates were used to measure fecal calprotectin and to perform 16S rRNA sequencing to characterize the gut microbiome. Fecal calprotectin was elevated in iCftr KO relative to WT samples of mice consuming either PEG or LiqD. PEG iCftr KO mice did not show a change in α-diversity versus WT but demonstrated a significant difference in microbial composition (ß-diversity) with increases in phylum Proteobacteria , family Peptostreptococcaceae , four genera of Clostridia including C. innocuum , and mucolytic genus Akkermansia . Fecal microbiome analysis of LiqD iCftr KO mice showed both decreased α-diversity and differences in microbial composition with increases in Proteobacteria family Enterobacteriaceae , Firmicutes families Clostridiaceae and Peptostreptococcaceae , and enrichment of Clostridium perfringens , C. innocuum , C. difficile , mucolytic Ruminococcus gnavus , and reduction of Akkermansia . It was concluded that epithelial-specific loss of Cftr is a major driver of CF intestinal dysbiosis and inflammation with significant similarities to previous studies of global Cftr KO mice. New and noteworthy: Chronic intestinal inflammation is a manifestation of cystic fibrosis (CF), a disease caused by loss of the anion channel CFTR that is expressed in many tissues. This study shows that intestinal epithelial cell-specific loss of CFTR (iCftr KO) in mice is sufficient to induce intestinal dysbiosis and inflammation. Studies were performed on mice consuming either dietary regimen (PEG laxative or liquid diet) routinely used to prevent obstruction in CF mice.

3.
Am J Physiol Gastrointest Liver Physiol ; 322(2): G282-G293, 2022 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-34878935

RESUMEN

Goblet cell hyperplasia is an important manifestation of cystic fibrosis (CF) disease in epithelial-lined organs. Explants of CF airway epithelium show normalization of goblet cell numbers; therefore, we hypothesized that small intestinal enteroids from Cftr knockout (KO) mice would not exhibit goblet cell hyperplasia. Toll-like receptors 2 and 4 (Tlr2 and Tlr4) were investigated as markers of inflammation and influence on goblet cell differentiation. Ex vivo studies found goblet cell hyperplasia in Cftr KO jejunum compared with wild-type (WT) mice. IL-13, SAM pointed domain-containing ETS transcription factor (Spdef), Tlr2, and Tlr4 protein expression were increased in Cftr KO intestine relative to WT. In contrast, WT and Cftr KO enteroids did not exhibit differences in basal or IL-13-stimulated goblet cell numbers, or differences in expression of Tlr2, Tlr4, and Spdef. Ileal goblet cell numbers in Cftr KO/Tlr4 KO and Cftr KO/Tlr2 KO mice were not different from Cftr KO mice, but enumeration was confounded by altered mucosal morphology. Treatment with Tlr4 agonist LPS did not affect goblet cell numbers in WT or Cftr KO enteroids, whereas the Tlr2 agonist Pam3Csk4 stimulated goblet cell hyperplasia in both genotypes. Pam3Csk4 stimulation of goblet cell numbers was associated with suppression of Notch1 and Neurog3 expression and upregulated determinants of goblet cell differentiation. We conclude that goblet cell hyperplasia and inflammation of the Cftr KO small intestine are not exhibited by enteroids, indicating that this manifestation of CF intestinal disease is not epithelial-automatous but secondary to the altered CF intestinal environment.NEW & NOTEWORTHY Studies of small intestinal organoids from cystic fibrosis (CF) mice show that goblet cell hyperplasia and increased Toll-like receptor 2/4 expression are not primary manifestations of the CF intestine. Intestinal goblet cell hyperplasia in the CF mice was not strongly altered by genetic ablation of Tlr2 and Tlr 4, but could be induced in both wild-type and CF intestinal organoids by a Tlr2-dependent suppression of Notch signaling.


Asunto(s)
Células Caliciformes/metabolismo , Hiperplasia/metabolismo , Inflamación/metabolismo , Intestinos/metabolismo , Animales , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Ratones Noqueados , Organoides/metabolismo , Transducción de Señal/fisiología
4.
Nanomedicine (Lond) ; 16(21): 1857-1872, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34282923

RESUMEN

The aim of this study was to investigate the distribution, tolerance, and anticancer and antiviral activity of Zn-based physiometacomposites (PMCs). Manganese, iron, nickel and cobalt-doped ZnO, ZnS or ZnSe were synthesized. Cell uptake, distribution into 3D culture and mice, and biochemical and chemotherapeutic activity were studied by fluorescence/bioluminescence, confocal microscopy, flow cytometry, viability, antitumor and virus titer assays. Luminescence and inductively coupled plasma mass spectrometry analysis showed that nanoparticle distribution was liver >spleen >kidney >lung >brain, without tissue or blood pathology. Photophysical characterization as ex vivo tissue probes and LL37 peptide, antisense oligomer or aptamer delivery targeting RAS/Ras binding domain (RBD) was investigated. Treatment at 25 µg/ml for 48 h showed ≥98-99% cell viability, 3D organoid uptake, 3-log inhibition of ß-Galactosidase and porcine reproductive respiratory virus infection. Data support the preclinical development of PMCs for imaging and delivery targeting cancer and infectious disease.


Asunto(s)
Antivirales , Nanopartículas , Animales , Antivirales/farmacología , Línea Celular Tumoral , Supervivencia Celular , Luminiscencia , Ratones , Porcinos , Zinc/farmacología
5.
Vet Immunol Immunopathol ; 203: 21-29, 2018 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-30243369

RESUMEN

Resveratrol is a polyphenol that is safe to administer to dogs and has immunomodulating properties. Canine in vitro work indicated that resveratrol spared polymorphonuclear cell (PMN) phagocytosis but reduced the robustness of PMN oxidative burst and resulted in a pro-inflammatory leukocyte cytokine profile. The objective of this study was to determine the short-term effect of resveratrol on the healthy canine innate immune system in vivo. The hypothesis was that resveratrol would spare phagocytosis, depress the vigor of PMN oxidative burst, and result in a proinflammatory stimulated leukocyte cytokine profile in vivo. In an open-label study, whole blood was collected from 12 healthy, adult client-owned dogs on day 0 and 3. Six dogs received resveratrol, 200 mg kg-1, orally once daily for three days and six dogs served as controls with no supplement administered. Phagocytosis, oxidative burst and pathogen associated molecular pathogen stimulated leukocyte production of tumor necrosis factor (TNF), interleukin (IL)-6, and IL-10 were measured. Results between days 0 and 3 were compared using two way repeated measures analysis of variance and Fisher least significant difference method. A P -value of < 0.05 was considered statistically significant. Resveratrol administration resulted in an increased number of Escherichia coli phagocytized by PMNs and decreased robustness of the oxidative burst reaction. Resveratrol also increased stimulated TNF and IL-6 production with no effect on IL-10. Resveratrol had differential effects on peripheral innate immune system function in dogs. Studies of resveratrol including tissue compartments and the adaptive immune system are indicated to determine if these immunologic effects may be beneficial in disease states.


Asunto(s)
Citocinas/metabolismo , Factores Inmunológicos/farmacología , Fagocitosis/efectos de los fármacos , Estallido Respiratorio/efectos de los fármacos , Estilbenos/farmacología , Animales , Perros , Femenino , Interleucina-10/sangre , Interleucina-6/sangre , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Masculino , Resveratrol , Factor de Necrosis Tumoral alfa/sangre
6.
Vet Immunol Immunopathol ; 163(3-4): 164-73, 2015 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-25579176

RESUMEN

INTRODUCTION: Resveratrol, a naturally-occurring phytophenol, has been shown to bolster immune surveillance and reverse immunosenescence in a dose dependent manner in rodents and humans. Although safety and pharmacokinetic studies have been completed in dogs, the immunomodulatory effects of resveratrol in dogs has not yet been investigated. The objective of this study was to determine the effect of resveratrol on canine innate immune system function in vitro. The hypothesis was that similar to other species, low concentrations of resveratrol would stimulate while high concentrations would depress innate immune system function. METHODS: Whole blood was collected from six healthy, adult, client-owned dogs and was incubated with resveratrol at final concentrations of 6000 ng ml(-1), 3000 ng ml(-1), 1000 ng ml(-1), or control solution for 4h. Following incubation, phagocytosis and oxidative burst were evaluated using flow cytometry, and LPS-, lipoteichoic acid (LTA) - and peptidoglycan (PG)-stimulated leukocyte production of TNF, IL-6, and IL-10 were measured using a canine specific multiplex assay. RESULTS: Phagocytosis was not altered by resveratrol at any concentration compared to control. However, while the number of PMNs capable of performing oxidative burst did not change, the robustness of the reaction following stimulation with Escherichia coli and PMA was reduced in a dose dependent manner. In addition, LPS-, LTA-, PG, and PBS-stimulated TNF production was increased following incubation with all concentrations of resveratrol compared to control, and this effect was dose dependent. LTA-stimulated IL-6 was increased with resveratrol compared to control. Furthermore, LTA-stimulated IL-10 was decreased with 6000 ng ml(-1) and 3000 ng ml(-1) concentrations of resveratrol and PG-stimulated IL-10 production was decreased with all concentrations of resveratrol compared to control. The LPS-, LTA-, and PG-stimulated TNF:IL-10 ratio was increased with 6000 ng ml(-1) of resveratrol compared to control and lower resveratrol concentrations. CONCLUSION: While resveratrol was sparing to PMN phagocytosis, it reduced the robustness of PMN oxidative burst. Resveratrol also increased pro-inflammatory and decreased anti-inflammatory leukocyte cytokine production capacity in vitro. These data suggest that resveratrol supplementation may depress oxidative burst reactions while promoting pro-inflammatory leukocyte cytokine production and decreasing anti-inflammatory cytokine production. Based on these findings, further in vivo study regarding the effects of resveratrol on PMN oxidative burst capability and leukocyte cytokine production capacity are indicated prior to routine supplementation.


Asunto(s)
Citocinas/metabolismo , Perros/sangre , Regulación de la Expresión Génica/efectos de los fármacos , Leucocitos/efectos de los fármacos , Estallido Respiratorio/efectos de los fármacos , Estilbenos/farmacología , Animales , Citocinas/genética , Regulación de la Expresión Génica/inmunología , Leucocitos/metabolismo , Fagocitosis , Resveratrol
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