Administration of Dietary Microalgae Ameliorates Intestinal Parameters, Improves Body Weight, and Reduces Thawing Loss of Fillets in Broiler Chickens: A Pilot Study

This pilot investigation aimed at studying the feasibility of using a low dose (0.2%) of dietary microalgae as a means of improving intestinal morphometry, body weight, and selected meat quality parameters in broilers. A total of 72 one-day-old ROSS 308 male chicks were randomly separated into four groups; three experimental pens in which the birds were fed with biomass from Tysochrysis lutea, Tetraselmis chuii, and Porphyridium cruentum over 30 days and a control group. T. chuii and P. cruentum had a positive effect with regard to body weight. In treated animals, duodenal and ileal sections showed characteristic tall and thin villi, with serrated surfaces and goblet cell differentiation. In both sections, values of the villus-height-to-crypt-depth ratio were increased by microalgae ingestion. The thawing weight loss of fillets was reduced in T. chuii-fed animals. The positive effects exerted by T. chuii and P. cruentum on intestinal architecture were associated with the improved body weight. Arguably, these outcomes exhibit the potential of using these species to enhance growth performance in broiler chickens by promoting gut homeostasis and thus nutrient absorption.

LKB1 deficiency upregulates RELM-α to drive airway goblet cell metaplasia

Targeting airway goblet cell metaplasia is a novel strategy that can potentially reduce the chronic obstructive pulmonary disease (COPD) symptoms. Tumor suppressor liver kinase B1 (LKB1) is an important regulator of the proliferation and differentiation of stem/progenitor cells. In this study, we report that LKB1 expression was downregulated in the lungs of patients with COPD and in those of cigarette smoke-exposed mice. Nkx2.1Cre; Lkb1f/f mice with conditional loss of Lkb1 in mouse lung epithelium displayed airway mucus hypersecretion and pulmonary macrophage infiltration. Single-cell transcriptomic analysis of the lung tissues from Nkx2.1Cre; Lkb1f/f mice further revealed that airway goblet cell differentiation was altered in the absence of LKB1. An organoid culture study demonstrated that Lkb1 deficiency in mouse airway (club) progenitor cells promoted the expression of FIZZ1/RELM-α, which drove airway goblet cell differentiation and pulmonary macrophage recruitment. Additionally, monocyte-derived macrophages in the lungs of Nkx2.1Cre; Lkb1f/f mice exhibited an alternatively activated M2 phenotype, while expressing RELM-α, which subsequently aggravated airway goblet cell metaplasia. Our findings suggest that the LKB1-mediated crosstalk between airway progenitor cells and macrophages regulates airway goblet cell metaplasia. Moreover, our data suggest that LKB1 agonists might serve as a potential therapeutic option to treat respiratory disorders associated with goblet cell metaplasia.

Goblet Cell Hyperplasia is not Epithelial-Autonomous in the Cftr Knockout Intestine

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, Tlr4) were investigated as markers of inflammation and influence on goblet cell differentiation. Ex vivo studies found goblet cell hyperplasia in Cftr KO jejunum as compared to wild-type (WT). IL-13, SAM pointed domain-containing ETS transcription factor (Spdef), Tlr2 and Tlr4 protein expression was 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.

Colorectal Cancer Progression Is Potently Reduced by a Glucose-Free, High-Protein Diet: Comparison to Anti-EGFR Therapy

To enable rapid proliferation, colorectal tumor cells up-regulate epidermal growth factor receptor (EGFR) signaling and aerobic glycolysis, resulting in substantial lactate release into the tumor microenvironment and impaired anti-tumor immune responses. We hypothesized that a nutritional intervention designed to reduce aerobic glycolysis may boost the EGFR-directed antibody (Ab)-based therapy of pre-existing colitis-driven colorectal carcinoma (CRC). CRC development was induced by azoxymethane (AOM) and dextran sodium sulfate (DSS) administration to C57BL/6 mice. AOM/DSS-treated mice were fed a glucose-free, high-protein diet (GFHPD) or an isoenergetic control diet (CD) in the presence or absence of an i.p. injection of an anti-EGFR mIgG2a or respective controls. AOM/DSS-treated mice on a GFHPD displayed a reduced systemic glucose metabolism associated with reduced oxidative phosphorylation (OXPHOS) complex IV expression and diminished tumor loads. Comparable but not additive to an anti-EGFR-Ab therapy, the GFHPD was accompanied by enhanced tumoral goblet cell differentiation and decreased colonic PD-L1 and splenic CD3e, as well as PD-1 immune checkpoint expression. In vitro, glucose-free, high-amino acid culture conditions reduced proliferation but improved goblet cell differentiation of murine and human CRC cell lines MC-38 and HT29-MTX in combination with down-regulation of PD-L1 expression. We here found GFHPD to systemically dampen glycolysis activity, thereby reducing CRC progression with a similar efficacy to EGFR-directed antibody therapy.

Mucus Release and Airway Constriction by TMEM16A May Worsen Pathology in Inflammatory Lung Disease

Activation of the Ca2+ activated Cl- channel TMEM16A is proposed as a treatment in inflammatory airway disease. It is assumed that activation of TMEM16A will induce electrolyte secretion, and thus reduce airway mucus plugging and improve mucociliary clearance. A benefit of activation of TMEM16A was shown in vitro and in studies in sheep, but others reported an increase in mucus production and airway contraction by activation of TMEM16A. We analyzed expression of TMEM16A in healthy and inflamed human and mouse airways and examined the consequences of activation or inhibition of TMEM16A in asthmatic mice. TMEM16A was found to be upregulated in the lungs of patients with asthma or cystic fibrosis, as well as in the airways of asthmatic mice. Activation or potentiation of TMEM16A by the compounds Eact or brevenal, respectively, induced acute mucus release from airway goblet cells and induced bronchoconstriction in mice in vivo. In contrast, niclosamide, an inhibitor of TMEM16A, blocked mucus production and mucus secretion in vivo and in vitro. Treatment of airway epithelial cells with niclosamide strongly inhibited expression of the essential transcription factor of Th2-dependent inflammation and goblet cell differentiation, SAM pointed domain-containing ETS-like factor (SPDEF). Activation of TMEM16A in people with inflammatory airway diseases is likely to induce mucus secretion along with airway constriction. In contrast, inhibitors of TMEM16A may suppress pulmonary Th2 inflammation, goblet cell metaplasia, mucus production, and bronchoconstriction, partially by inhibiting expression of SPDEF.

EHF is essential for epidermal and colonic epithelial homeostasis, and suppresses Apc-initiated colonic tumorigenesis

ABSTRACT Ets homologous factor (EHF) is a member of the epithelial-specific Ets (ESE) family of transcription factors. To investigate its role in development and epithelial homeostasis, we generated a series of novel mouse strains in which the Ets DNA-binding domain of Ehf was deleted in all tissues (Ehf−/−) or specifically in the gut epithelium. Ehf−/− mice were born at the expected Mendelian ratio, but showed reduced body weight gain, and developed a series of pathologies requiring most Ehf−/− mice to reach an ethical endpoint before reaching 1 year of age. These included papillomas in the facial skin, abscesses in the preputial glands (males) or vulvae (females), and corneal ulcers. Ehf−/−mice also displayed increased susceptibility to experimentally induced colitis, which was confirmed in intestinal-specific Ehf knockout mice. Gut-specific Ehf deletion also impaired goblet cell differentiation, induced extensive transcriptional reprogramming in the colonic epithelium and enhanced Apc-initiated adenoma development. The Ets DNA-binding domain of EHF is therefore essential for postnatal homeostasis of the epidermis and colonic epithelium, and its loss promotes colonic tumour development.

Thyroid hormones regulate goblet cell differentiation and Fgf19-Fgfr4 signaling

Hypothyroidism is a common pathological condition that is characterized by insufficient activity of the thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3), in the whole body or in specific tissues. Hypothyroidism is associated with inadequate development of the intestine as well as gastrointestinal diseases. We utilized a zebrafish model of hypothyroidism in order to identify and characterize TH-modulated genes and cellular pathways controlling intestine development. In the intestine of hypothyroid juveniles and adults, the number of mucus-secreting goblet cells was reduced, and this phenotype could be rescued by T3 treatment. Transcriptome profiling revealed dozens of differentially expressed genes in the intestine of hypothyroid adults compared to controls. Notably, the expression of genes encoding to Fgf19 and its receptor Fgfr4 was markedly increased in the intestine of hypothyroid adults, and treatment with T3 normalized it. Blocking FGF signaling, using an inducible dominant negative Fgfr transgenic line, rescued the number of goblet cells in hypothyroid adults. These results show that THs inhibit the Fgf19-Fgfr4 signaling pathway, which is associated with inhibition of goblet cell differentiation in hypothyroidism. Both the TH and Fgf19-Fgfr4 signaling pathways can be pharmaceutical targets for the treatment of TH-related gastrointestinal diseases.

Dynamic association of IκBα to chromatin is regulated by acetylation and cleavage of histone H4

ABSTRACTIκBs exert a principal function as cytoplasmic inhibitors of the NF-kB transcription factors. Additional functions for IκB homologues have been described including association to chromatin and transcriptional regulatioin. Phosphorylated and SUMOylated IκBα (pS-IκBα) binds histones H2A and H4 in the stem and progenitor compartment of skin and intestine, but the mechanisms controlling its recruitment to chromatin are largely unstudied.We here show that serine 32-36 phosphorylation of IκBα favors its binding with nucleosomes and demonstrated that p-IκBα association to H4 is favored by acetylation at specific H4 lysine residues. N-terminal tail of H4 is lost during intestinal cell differentiation by proteolytic cleavage at residues 17-19 imposed ny trypsin or chymotrypsin, which interferes p-IκBα binding. Paradoxically, inhibition of trypsin and chymotrypsin activity in HT29 cells increased p-IκBα chromatin binding and impaired goblet cell differentiation, comparable to IκBα deletion. Together our results indicate that dynamic binding of IκBα to chromatin is a requirement for intestinal cell differentiation and provide a molecular base for the restricted nuclear distribution of p-IκBα at specific stem cell compartments.