Constitutive Differential Features of Type 2 Transglutaminase in Cells Derived from Celiac Patients and from Healthy Subjects

Type 2 transglutaminase (TG2) is a ubiquitous enzyme able to modify gliadin peptides introduced into the organism through the diet. By means of its catalytic activity, TG2 seems to have an important pathogenetic role in celiac disease (CD), an inflammatory intestinal disease caused by the ingestion of gluten-containing cereals. A strong autoimmune response to TG2 characterizes CD development. Anti-TG2 antibodies specifically derange the uptake of the α-gliadin peptide 31-43 by control, but not by celiac dermal fibroblasts, underlying some different constitutive features regarding TG2 in healthy and celiac subjects. Our aim was to investigate whether these differences depended on a different TG2 subcellular distribution and whether peptide 31-43 differentially regulated TG2 expression and activity in cells of the two groups of subjects. We found that TG2 was more abundantly associated with membranes of celiac fibroblasts than of control cells, in particular with the early endosomal and autophagic compartments. We also found that peptide 31-43 differentially affected TG2 expression and activity in the two groups of cells, activating TG2 more in control than in celiac cells and inducing TG2 expression in celiac cells, but not in control ones. The different TG2 subcellular localization and the different way the peptide 31-43 modulates TG2 activity and availability into control and CD cells suggested that TG2 is involved in the definition of a constitutive CD cellular phenotype, thus having an important and still undefined role in CD pathogenesis

Enterocyte Proliferation and Signaling Are Constitutively Altered in Celiac Disease

Celiac disease (CD) occurs frequently, and is caused by ingestion of prolamins from cereals in subjects with a genetic predisposition. The small intestinal damage depends on an intestinal stress/innate immune response to certain gliadin peptides (e.g., A-gliadin P31-43) in association with an adaptive immune response to other gliadin peptides (e.g., A-gliadin P57-68). Gliadin and peptide P31-43 affect epithelial growth factor receptor (EGFR) signaling and CD enterocyte proliferation. The reason why the stress/innate immune and proliferative responses to certain gliadin peptides are present in CD and not in control intestine is so far unknown. The aim of this work is to investigate if, in CD, a constitutive alteration of enterocyte proliferation and signaling exists that may represent a predisposing condition to the damaging effects of gliadin. Immunofluorescence and immunohistochemistry were used to study signaling in CD fibroblasts and intestinal biopsies. Western blot (WB) analysis, immunoprecipitation, and quantitative PCR were also used. We found in CD enterocytes enhancement of both proliferation and Epidermal Growth Factor Receptor (EGFR)/ligand system. In CD enterocytes and fibroblasts we found increase of the phosphorylated downstream signaling molecule Extracellular Signal Regulated Kinase (ERK); block of the ERK activation normalizes enterocytes proliferation in CD mucosa. In conclusion the same pathway, which gliadin and gliadin peptide P31-43 can interfere with, is constitutively altered in CD cells. This observation potentially explains the specificity of the damaging effects of certain gliadin peptides on CD intestine.</p

Celiac disease-associated Neisseria flavescens decreases mitochondrial respiration in CaCo-2 epithelial cells: Impact of Lactobacillus paracasei CBA L74 on bacterial-induced cellular imbalance

: We previously identified a Neisseria flavescens strain in the duodenum of celiac disease (CD) patients that induced immune inflammation in ex vivo duodenal mucosal explants and in CaCo-2 cells. We also found that vesicular trafficking was delayed after the CD-immunogenic P31-43 gliadin peptide-entered CaCo-2 cells and that Lactobacillus paracasei CBA L74 (L.&nbsp;paracasei-CBA) supernatant reduced peptide entry. In this study, we evaluated if metabolism and trafficking was altered in CD-N.&nbsp;flavescens-infected CaCo-2 cells and if any alteration could be mitigated by pretreating cells with L.&nbsp;paracasei-CBA supernatant, despite the presence of P31-43. We measured CaCo-2 bioenergetics by an extracellular flux analyser, N.&nbsp;flavescens and P31-43 intracellular trafficking by immunofluorescence, cellular stress by TBARS assay, and ATP by bioluminescence. We found that CD-N. flavescens colocalised more than control N.&nbsp;flavescens with early endocytic vesicles and more escaped autophagy thereby surviving longer in infected cells. P31-43 increased colocalisation of N.&nbsp;flavescens with early vesicles. Mitochondrial respiration was lower (P&nbsp;&lt;&nbsp;.05) in CD-N.&nbsp;flavescens-infected cells versus not-treated CaCo-2 cells, whereas pretreatment with L.&nbsp;paracasei-CBA reduced CD-N.&nbsp;flavescens viability and improved cell bioenergetics and trafficking. In conclusion, CD-N.&nbsp;flavescens induces metabolic imbalance in CaCo-2 cells, and the L.&nbsp;paracasei-CBA probiotic could be used to correct CD-associated dysbiosis

Gliadin-Mediated Proliferation and Innate Immune Activation in Celiac Disease Are Due to Alterations in Vesicular Trafficking

Background and Objectives: Damage to intestinal mucosa in celiac disease (CD) is mediated both by inflammation due to adaptive and innate immune responses, with IL-15 as a major mediator of the innate immune response, and by proliferation of crypt enterocytes as an early alteration of CD mucosa causing crypts hyperplasia. We have previously shown that gliadin peptide P31-43 induces proliferation of cell lines and celiac enterocytes by delaying degradation of the active epidermal growth factor receptor (EGFR) due to delayed maturation of endocytic vesicles. IL-15 is increased in the intestine of patients affected by CD and has pleiotropic activity that ultimately results in immunoregulatory cross-talk between cells belonging to the innate and adaptive branches of the immune response. Aims of this study were to investigate the role of P31-43 in the induction of cellular proliferation and innate immune activation. Methods/Principal Findings: Cell proliferation was evaluated by bromodeoxyuridine (BrdU) incorporation both in CaCo-2 cells and in biopsies from active CD cases and controls. We used real-time PCR to evaluate IL-15 mRNA levels and FACS as well as ELISA and Western Blot (WB) analysis to measure protein levels and distribution in CaCo-2 cells. Gliadin and P31-43 induce a proliferation of both CaCo-2 cells and CD crypt enterocytes that is dependent on both EGFR and IL-15 activity. In CaCo-2 cells, P31-43 increased IL-15 levels on the cell surface by altering intracellular trafficking. The increased IL-15 protein was bound to IL15 receptor (IL-15R) alpha, did not require new protein synthesis and functioned as a growth factor. Conclusion: In this study, we have shown that P31-43 induces both increase of the trans-presented IL-15/IL5R alpha complex on cell surfaces by altering the trafficking of the vesicular compartments as well as proliferation of crypt enterocytes with consequent remodelling of CD mucosa due to a cooperation of IL-15 and EGFR

The protective role of Lactobacillus rhamnosus GG postbiotic on the alteration of autophagy and inflammation pathways induced by gliadin in intestinal models

Celiac disease (CD) is an autoimmune enteropathy caused by an abnormal immune response to gliadin peptides in genetically predisposed individuals. For people with CD, the only available therapy thus far is the lifelong necessity for a gluten-free diet (GFD). Innovative therapies include probiotics and postbiotics as dietary supplements, both of which may benefit the host. Therefore, the present study aimed to investigate the possible beneficial effects of the postbiotic Lactobacillus rhamnosus GG (LGG) in preventing the effects induced by indigested gliadin peptides on the intestinal epithelium. In this study, these effects on the mTOR pathway, autophagic function, and inflammation have been evaluated. Furthermore, in this study, we stimulated the Caco-2 cells with the undigested gliadin peptide (P31-43) and with the crude gliadin peptic-tryptic peptides (PTG) and pretreated the samples with LGG postbiotics (ATCC 53103) (1 × 108). In this study, the effects induced by gliadin before and after pretreatment have also been investigated. The phosphorylation levels of mTOR, p70S6K, and p4EBP-1 were increased after treatment with PTG and P31-43, indicating that the intestinal epithelial cells responded to the gliadin peptides by activating the mTOR pathway. Moreover, in this study, an increase in the phosphorylation of NF-κβ was observed. Pretreatment with LGG postbiotic prevented both the activation of the mTOR pathway and the NF-κβ phosphorylation. In addition, P31-43 reduced LC3II staining, and the postbiotic treatment was able to prevent this reduction. Subsequently, to evaluate the inflammation in a more complex intestinal model, the intestinal organoids derived from celiac disease patient biopsies (GCD-CD) and controls (CTR) were cultured. Stimulation with peptide 31-43 in the CD intestinal organoids induced NF-κβ activation, and pretreatment with LGG postbiotic could prevent it. These data showed that the LGG postbiotic can prevent the P31-43-mediated increase in inflammation in both Caco-2 cells and in intestinal organoids derived from CD patients

Early results from GLASS-JWST. XIII. A faint, distant, and cold brown dwarf

We present the serendipitous discovery of a late T-type brown dwarf candidate in JWST NIRCam observations of the Early Release Science Abell 2744 parallel field. The discovery was enabled by the sensitivity of JWST at 4~$\mu$m wavelengths and the panchromatic 0.9--4.5~$\mu$m coverage of the spectral energy distribution. The unresolved point source has magnitudes F115W = 27.95$\pm$0.15 and F444W = 25.84$\pm$0.01 (AB), and its F115W$-$F444W and F356W$-$F444W colors match those expected for other, known T dwarfs. We can exclude it as a reddened background star, high redshift quasar, or very high redshift galaxy. Comparison with stellar atmospheric models indicates a temperature of $T_{eff}$ $\approx$ 600~K and surface gravity $\log{g}$ $\approx$ 5, implying a mass of 0.03~M$_{\odot}$ and age of 5~Gyr. We estimate the distance of this candidate to be 570--720~pc in a direction perpendicular to the Galactic plane, making it a likely thick disk or halo brown dwarf. These observations underscore the power of JWST to probe the very low-mass end of the substellar mass function in the Galactic thick disk and halo.Comment: ApJL accepte

Early Results From GLASS-JWST. XVII: Building the First Galaxies -- Chapter 1. Star Formation Histories at 5<z < 7

JWST observations of high redshift galaxies are used to measure their star formation histories - the buildup of stellar mass in the earliest galaxies. Here we use a novel analysis program, SEDz*, to compare near-IR spectral energy distributions for galaxies with redshifts 5 < z < 7 to combinations of stellar population templates evolved from z = 12. We exploit NIRCam imaging in 7 wide bands covering 1-5 mu m, taken in the context of the GLASS-JWST-ERS program, and use SEDz* to solve for well-constrained star formation histories for 24 exemplary galaxies. In this first look we find a variety of histories, from long, continuous star formation over 5 < z < 12 to short but intense starbursts - sometimes repeating, and, most commonly, contiguous mass buildup lasting ~ 0.5 Myr,possibly the seeds of today's typical, M* galaxies.Comment: ApJL in press (accepted on October 30, 2022

Early results from GLASS-JWST XV: properties of the faintest red sources in the NIRCAM deep fields

We present a first look at the reddest 2-5$\mu\rm m$ sources found in deep images from the GLASS Early Release Science program. We undertake a general search, i.e. not looking for any particular spectral signatures, for sources detected only in bands redder than reachable with the Hubble Space Telescope, and which would likely not have been identified in pre-JWST surveys. We search for sources down to AB $\sim 27$ (corresponding to $>10\sigma$ detection threshold) in any of the F200W to F444W filters,with a $>1$ magnitude excess relative to F090W to F150W bands. Fainter than F444W$>25$ we find 56 such sources of which 37 have reasonably constrained spectral energy distributions to which we can fit photometric redshifts. We find the majority of this population ($\sim$ 65%) as $2<z<6$ star forming low-attenuation galaxies that are faint at rest-frame ultraviolet-optical wavelengths, have stellar masses $10^{8.5}$-$10^{9.5}$M$_\odot$, and have observed fluxes at $>$2$\mu \rm m$ boosted by a combination of the Balmer break and emission lines. The typical implied rest equivalent widths are \sim200\unicode{0x212B} with some extreme objects up to \sim 1000\unicode{0x212B}. This is in contrast with brighter magnitudes where the red sources tend to be $z<3$ quiescent galaxies and dusty star forming objects. Our general selection criteria for red sources allow us to independently identify other phenomena as diverse as extremely low mass ($\sim 10^8$ M$_\odot$) quiescent galaxies at $z<1$, recover recently identified $z>11$ galaxies and a very cool brown dwarf.Comment: Accepted for publication in Astrophysical Journal Letters. 11 pages, 3 figures. Updated with post-flight JWST NIRCAM calibrations leading to significantly revised conclusions. V1 should be discounte

Early results from GLASS-JWST XVI: Discovering a bluer z~4-7 Universe through UV slopes

We use the GLASS-JWST Early Release Science NIRCam parallel observations to provide a first view of the UV continuum properties of NIRCam/F444W selected galaxies at 4<z<7. By combining multiwavelength NIRCam observations, we constrain the UV continuum slope for a sample of 401 galaxies with stringent quality controls. We find that >99% of the galaxies are blue star-forming galaxies with very low levels of dust (Avbeta~0.01+/-0.33). We find no statistically significant correlation for UV slope with redshift or UV magnitude. However, we find that in general galaxies at higher redshifts and fainter UV magnitudes have steeper UV slopes. We find a statistically significant correlation for UV slope with stellar mass, with galaxies with higher stellar mass showing shallower UV slopes. Individual fits to some of our galaxies reach the bluest UV slopes of beta~-3.1 allowed by stellar population models used in this analysis. Therefore, it is likely that stellar population models with higher amount of Lyman continuum leakage, AGN effects, and/or Population III contributions are required to accurately reproduce the rest-UV and optical properties of some of our bluest galaxies. This dust-free early view confirms that our current cosmological understanding of gradual mass + dust buildup of galaxies with cosmic time is largely accurate to describe the ~0.7-1.5 Gyr age window of the Universe. The abundance of a large population of UV faint dust-poor systems may point to a dominance of low-mass galaxies at z>6 playing a vital role in cosmic reionization.Comment: Accepted in ApJ

Early Results from GLASS-JWST. XXI: Rapid assembly of a galaxy at z=6.23 revealed by its C/O abundance

The abundance of carbon relative to oxygen (C/O) is a promising probe of star formation history in the early universe, as the ratio changes with time due to production of these elements by different nucleosynthesis pathways. We present a measurement of $\log{\mathrm{(C/O)}} = -1.01\pm0.12$ (stat) $\pm0.15$ (sys) in a $z=6.23$ galaxy observed as part of the GLASS-JWST Early Release Science Program. Notably, we achieve good precision thanks to the detection of the rest-frame ultraviolet O III], C III], and C IV emission lines delivered by JWST/NIRSpec. The C/O abundance is $\sim$0.8 dex lower than the solar value and is consistent with the expected yield from core-collapse supernovae, indicating that longer-lived intermediate mass stars have not fully contributed to carbon enrichment. This in turn implies rapid buildup of a young stellar population with age $\lesssim100$ Myr in a galaxy seen $\sim$900 million years after the Big Bang. Our chemical abundance analysis is consistent with spectral energy distribution modeling of JWST/NIRCam photometric data, which indicates a current stellar mass $\log\,\mathrm{M}_* / \mathrm{M_{sun}} = 8.4^{+0.4}_{-0.2}$ and specific star formation rate sSFR $\simeq 20$ Gyr$^{-1}$. These results showcase the value of chemical abundances and C/O in particular to study the earliest stages of galaxy assembly.Comment: 16 pages, 4 figures, 2 tables. Accepted for publication in ApJ