Abstract
Background and Aims
Endoplasmic reticulum ER stress in intestinal epithelial cells IECs contributes to the pathogenesis of inflammatory bowel disease IBD. We hypothesized that ER stress ...changes innate signalling in human IECs, augmenting toll-like receptor TLR responses and inducing pro-inflammatory changes in underlying dendritic cells DCs.
Methods
Caco-2 cells and primary human colon-derived enteroid monolayers were exposed to ATP control stressor or thapsigargin Tg ER stress inducer, and were stimulated with the TLR5 agonist flagellin. Cytokine release was measured by an enzyme immunoassay. ER stress markers CHOP, GRP78 and XBP1s/u were measured via quantitative PCR and Western blot. Monocyte-derived DCs moDCs were cultured with the IEC supernatants and their activation state was measured. Responses from enteroids derived from IBD patients and healthy control participants were compared.
Results
ER stress enhanced flagellin-induced IL-8 release from Caco-2 cells and enteroids. Moreover, conditioned media activated DCs to become pro-inflammatory, with increased expression of CD80, CD86, MHCII, IL-6, IL-15 and IL-12p70 and decreased expression of CD103 and IL-10. Flagellin-induced IL-8 production correlated with DC activation, suggesting a common stress pathway. Moreover, there were distinct differences in cytokine expression and basal ER stress between IBD and healthy subject-derived enteroid monolayers, suggesting a dysregulated ER stress pathway in IBD-derived enteroids.
Conclusions
Cellular stress enhances TLR5 responses in IECs, leading to increased DC activation, indicating a previously unknown mechanistic link between epithelial ER stress and immune activation in IBD. Furthermore, dysregulated ER stress may be propagated from the intestinal epithelial stem cell niche in IBD patients.
Regulatory T cell (Treg) therapy is a promising strategy to treat inflammatory bowel disease (IBD). Data from animal models has shown that Tregs specific for intestinal antigens are more potent than ...polyclonal Tregs at inhibiting colitis. Flagellins, the major structural proteins of bacterial flagella, are immunogenic antigens frequently targeted in IBD subjects, leading to the hypothesis that flagellin-specific Tregs could be an effective cell therapy for IBD. We developed a novel chimeric antigen receptor (CAR) specific for flagellin derived from Escherichia coli H18 (FliC). We used this CAR to confer FliC-specificity to human Tregs and investigated their therapeutic potential. FliC-CAR Tregs were activated by recombinant FliC protein but not a control flagellin protein, demonstrating CAR specificity and functionality. In a humanized mouse model, expression of the FliC-CAR drove preferential migration to the colon and expression of the activation marker PD1. In the presence of recombinant FliC protein in vitro, FliC-CAR Tregs were significantly more suppressive than control Tregs and promoted the establishment of colon-derived epithelial cell monolayers. These results demonstrate the potential of FliC-CAR Tregs to treat IBD and more broadly show the therapeutic potential of CARs targeting microbial-derived antigens.
•Chimeric antigen receptors (CAR) can be used to confer microbe specificity to Tregs.•Flagellin-specific CAR Tregs preferentially home to damaged intestinal tissues.•CAR Tregs elicit antigen-dependent immunosuppression.•Activated CAR Tregs secrete factors that promote intestinal barrier integrity.
Clostridioides difficile is a toxin-secreting bacteria that is an urgent antimicrobial resistance threat, with approximately 25% of patients developing recurrent infections. Inflammatory bowel ...disease (IBD) patients are at increased risk of severe, recurrent C. difficile infection.
To investigate a role for C. difficile infection in IBD pathogenesis, we collected peripheral blood and stool from 20 each of ulcerative colitis patients, Crohn's disease patients, and healthy control subjects. We used a flow cytometric activation induced marker assay to quantify C. difficile toxin-specific CD4+ T cells and 16S ribosomal RNA sequencing to study microbiome diversity.
We found IBD patients had significantly increased levels of C. difficile toxin B-specific CD4+ T cells, but not immunoglobulin G or immunoglobulin A, compared with healthy control subjects. Within antigen-specific CD4+ T cells, T helper type 17 cells and cells expressing the gut homing receptor integrin β7 were reduced compared with healthy control subjects, similar to our previous study of non-IBD patients with recurrent C. difficile infection. Stool microbiome analysis revealed that gut homing, toxin-specific CD4+ T cells negatively associated with microbial diversity and, along with T helper type 17 cells, positively associated with bacteria enriched in healthy control subjects.
These data suggest that IBD patients, potentially due to underlying intestinal dysbiosis, experience undiagnosed C. difficile infections that result in impaired toxin-specific immunity. This may contribute to the development of inflammatory T cell responses toward commensal bacteria and provide a rationale for C. difficile testing in IBD patients.
Objective: To investigate whether catch‐up growth after maternal malnutrition would favor the development of obesity in adulthood.
Research Methods and Procedures: Pregnant rats were submitted to ...protein or calorie restriction during the course of gestation. During lactation, pups were protein‐restricted, normally fed, or overfed reduced litter size, control (C) diet. At weaning, rats were transferred to chow or to a hypercaloric diet (HCD) known to induce obesity. Body weight, food intake, blood parameters, glucose tolerance, adipocyte cellularity, and adipose factors contributing to cardiovascular disease development were measured.
Results: Protein and calorie restriction during gestation led to growth retardation at birth. If malnutrition was prolonged throughout lactation, adult body weight was permanently reduced. However, growth‐retarded offspring overfed during the suckling period underwent a rapid catch‐up growth and became heavier than the normally fed Cs. Offspring of calorie‐restricted rats gained more weight than those of dams fed protein‐restricted diet. Feeding an HCD postnatally amplified the effect of calorie restriction, and offspring that underwent catch‐up growth became more obese than Cs. The HCD was associated with hyperphagia, hyperglycemia, hyperinsulinemia, glucose intolerance, insulin resistance, and adipocyte hypertrophy. The magnitude of effects varied depending on the type and the timing of early malnutrition. The expression of genes encoding factors implicated in cardiovascular disease was also modulated differently by early malnutrition and adult obesity.
Discussion: Catch‐up growth immediately after early malnutrition should be a key point for the programming of obesity.
Controlled ion bombardment of growing thin films can be used to modify and improve the film structure and properties. Recently, higher energetic species (up to hundreds eV) were found in the plasma ...by pulsing the target(s) in magnetron sputtering. In this study, an electrostatic quadrupole plasma mass spectrometer (EQP) has been used in a pulsed closed unbalanced magnetron sputtering (P-CFUBMS) system to investigate the effect of different pulsing parameters (frequency and reverse time) on the ion energies and ion fluxes in the intrinsic plasma during Cr–Al–N film deposition. It is confirmed that pulsing both magnetrons in this P-CFUBMS configuration had a large effect on both the ion energies and ion fluxes generated within the plasma, which are shown to be strongly dependent on pulsing frequency and duty cycle.
The effect of pulsing to provide a wide range of ion energies and ion fluxes on the film microstructure, mechanical and tribological properties was investigated using nanoindentation, microtribometry, X-ray diffraction (XRD), atomic force microscopy (AFM), field emission scanning electron microscopy (FESEM) and scanning transmission electron microscopy (TEM). In the current study, by taking −50 V substrate bias into consideration, it was found that total ion energies with controlled pulsing parameters to achieve moderate values (70–120 eV) can effectively increase the density and decrease the grain size of Cr–Al–N films. On the other hand, pulsing regimes that produce excessive total ion energy (∼
200 eV) result in an increase in the residual strain, and point and lattice defects in the film, which will significantly decrease the toughness and tribological properties of the film. Under optimum pulsing conditions (100 kHz and 5.0 μs), Cr–Al–N films with a dense nanostructure (column grain size of 10–40 nm) of super hardness and good wear resistance (41 GPa, 0.099 H/E ratio, 0.46 COF, and a wear rate of 3.4
×
10
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3N
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) have been deposited using a controlled maximum ion energy bombardment of 122 eV at high ion flux.
Acute damage to the intestinal epithelium can be repaired via de-differentiation of mature intestinal epithelial cells (IECs) to a stem-like state, but there is a lack of knowledge on how intestinal ...stem cells function after chronic injury, such as in inflammatory bowel disease (IBD). We developed a chronic-injury model in human colonoid monolayers by repeated rounds of air-liquid interface and submerged culture. We use this model to understand how chronic intestinal damage affects the ability of IECs to (1) respond to microbial stimulation, using the Toll-like receptor 5 (TLR5) agonist FliC and (2) regenerate and protect the epithelium from further damage. Repeated rounds of damage impair the ability of IECs to regrow and respond to TLR stimulation. We also identify mRNA expression and DNA methylation changes in genes associated with IBD and colon cancer. This methodology results in a human model of recurrent IEC injury like that which occurs in IBD.
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•Repeated rounds of air-liquid interface growth and submergence damage human colonoids•Colonoids lose regenerative capacity with repeated injury•Repeated injury causes loss of inflammatory responsiveness to flagellin•Repeated injury causes changes in gene expression and DNA methylation resembling IBD
Rees et al. develop a model of recurrent injury to human colonoids by alternating growth in air-liquid interface and submerged culture. This injury causes loss of barrier integrity and regrowth potential and gene expression and global DNA methylation changes that resemble those seen in inflammatory bowel disease and colon cancer.
Many developmental syndromes have been linked to genetic mutations that cause abnormal ERK/MAPK activity; however, the neuropathological effects of hyperactive signaling are not fully understood. ...Here, we examined whether hyperactivation of MEK1 modifies the development of GABAergic cortical interneurons (CINs), a heterogeneous population of inhibitory neurons necessary for cortical function. We show that GABAergic-neuron specific MEK1 hyperactivation in vivo leads to increased cleaved caspase-3 labeling in a subpopulation of immature neurons in the embryonic subpallial mantle zone. Adult mutants displayed a significant loss of parvalbumin (PV), but not somatostatin, expressing CINs and a reduction in perisomatic inhibitory synapses on excitatory neurons. Surviving mutant PV-CINs maintained a typical fast-spiking phenotype but showed signs of decreased intrinsic excitability that coincided with an increased risk of seizure-like phenotypes. In contrast to other mouse models of PV-CIN loss, we discovered a robust increase in the accumulation of perineuronal nets, an extracellular structure thought to restrict plasticity. Indeed, we found that mutants exhibited a significant impairment in the acquisition of behavioral response inhibition capacity. Overall, our data suggest PV-CIN development is particularly sensitive to hyperactive MEK1 signaling, which may underlie certain neurological deficits frequently observed in ERK/MAPK-linked syndromes.
Background: Mycobacterium avium subspecies paratuberculosis (MAP), a member of the mycobacteriaceae family, causes Johne’s disease in ruminants, which resembles Crohn’s disease (CD) in humans. MAP ...was proposed to be one of the causes of human CD, but the evidence remains elusive. Macrophages were reported to be the only cell where MAP proliferates in ruminants and humans and is likely the major producer of TNFα-associated inflammation. However, whether human dendritic cells (DCs), another major antigen-presenting cell (APC), have the ability to harbor MAP and disseminate infection, remains unknown. Methods: Human monocyte-derived dendritic cells (moDCs) were infected with MAP and phagocytosis and intracellular survival were quantified by immunofluorescence (IF) and colony counts, respectively. MoDC cytokine expression was measured via ELISA and their activation state was measured via flow cytometry. Results: We showed that MAP can infect and replicate in human moDCs as means to evade the immune system for successful infection, through inhibition of the phago-lysosome fusion via the secretion of protein tyrosine phosphatase PtpA. This mechanism initially led to a state of tolerance in moDCs and then subsequently caused a pro-inflammatory response as infection persisted, characterized by the upregulation of IL-6 and TNFα, and downregulation of IL-10. Moreover, we showed that moDCs have the ability to phagocytose up to 18% of MAP, when exposed at a multiplicity of infection of 1:1. Conclusion: Infection and subsequent proliferation of MAP within moDCs could provide a unique means for the dissemination of MAP to lymphoid tissue, while altering immune responses to facilitate the persistence of infection of host tissues in CD.
Unsedated office-based laser surgery (UOLS) of the larynx and trachea has significantly improved the treatment options for patients with laryngotracheal pathology including recurrent respiratory ...papillomas, granulomas, leukoplakia, and polypoid degeneration. UOLS delivered by flexible endoscopes has dramatically impacted office-based surgery by reducing the time, costs, and morbidity of surgery.
To review our experience with 443 laryngotracheal cases treated by UOLS.
The laser logbooks at the Center for Voice and Swallowing Disorders were reviewed for UOLS, and the medical and laryngological histories were detailed, as were the treatment modalities, frequencies, and complications.
Of the 443 cases, 406 were performed with the pulsed-dye laser, 10 with the carbon-dioxide laser, and 27 with the thulium: yttrium-aluminum-garnet laser. There were no significant complications in this series. A review of indications and wavelength selection criteria is presented.
Unsedated, office-based, upper aerodigestive tract laser surgery appears to be a safe and effective treatment option for many patients with laryngotracheal pathology.
Tissue-clearing methods allow every cell in the mouse brain to be imaged without physical sectioning. However, the computational tools currently available for cell quantification in cleared tissue ...images have been limited to counting sparse cell populations in stereotypical mice. Here, we introduce NuMorph, a group of analysis tools to quantify all nuclei and nuclear markers within the mouse cortex after clearing and imaging by light-sheet microscopy. We apply NuMorph to investigate two distinct mouse models: a Topoisomerase 1 (Top1) model with severe neurodegenerative deficits and a Neurofibromin 1 (Nf1) model with a more subtle brain overgrowth phenotype. In each case, we identify differential effects of gene deletion on individual cell-type counts and distribution across cortical regions that manifest as alterations of gross brain morphology. These results underline the value of whole-brain imaging approaches, and the tools are widely applicable for studying brain structure phenotypes at cellular resolution.
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•NuMorph accurately quantifies all cortical nuclei and cell-type-specific markers•Top1 deletion results in increased neuronal degeneration in frontal areas•Nf1 deletion leads to structural changes that recapitulate human MRI findings•Structural changes are driven by increased gliogenesis in the Nf1 knockout model
Krupa et al. develop an image analysis toolbox called NuMorph that accurately quantifies all nuclei within the mouse cortex after tissue clearing and light-sheet imaging. They implement NuMorph to characterize region- and cell-type-specific deficits present in two distinct mouse models, demonstrating the advantages of a whole-brain imaging approach.
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