an-intestinal-gut-organ-culture-system-for-analyzing-host-microbiota

An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for ...

Bar Ilan University

Bar-Ilan University

The Aspergillus NIMA serine/threonine kinase plays a pivotal role in controlling entrance into mitosis. A major function attributed to NIMA is the induction of chromatin condensation. We show here that the founder murine NIMA-related kinase, Nek1, is larger than previously reported, and that the full-length protein conserves the structural hallmarks of NIMA. Even though Nek1 bears two classical nuclear localization signals (NLS), the endogenous protein localizes to the cytoplasm. Ectopic overexpression of various Nek1 constructs suggests that the C-terminus of Nek1 bears cytoplasmic localization signal(s). Overexpression of nuclear constructs of Nek1 resulted in abnormal chromatin condensation, with the DNA mainly confined to the periphery of the nucleus. Advanced condensation phenotype was associated with nuclear pore complex dispersal. The condensation was not accompanied by up-regulation of mitotic or apoptotic markers. A similar phenotype has been described following NIMA overexpression, strengthening the notion that the mammalian Nek1 kinase has functional similarity to NIMA.

Nek1 shares structural and functional similarities with NIMA kinase.

Members of the NIMA-related kinases (NRK) family are recently emerging as central regulators of various aspects of the cell cycle. However, the cellular roles of the mammalian NRK, Nek7, remain obscure. We show here that the endogenous Nek7 protein is enriched at the centrosome in a microtubule-independent manner. Overexpression of wt or kinase-defective Nek7 resulted in cells of rounder appearance, and higher proportions of multinuclear and apoptotic cells. Down-regulation of Nek7 using a small interfering RNA approach resulted in a significant increase in mitotic cells presenting multipolar spindle phenotype. These results suggest a role for Nek7 in regulating proper spindle assembly and mitotic progression.

Nek7 kinase is enriched at the centrosome, and is required for proper spindle assembly and mitotic progression.

Congenital dyserythropoietic anemia type I is an inherited autosomal recessive macrocytic anemia associated with ineffective erythropoiesis and the development of secondary hemochromatosis. Distinct erythroid precursors with internuclear chromatin bridges and spongy heterochromatin are pathognomonic for the disease. The mutated gene (CDAN1) encodes a ubiquitously expressed protein of unknown function, codanin-1. Based on the morphological features of congenital dyserythropoietic anemia type I erythroblasts and data on a role in cell cycle progression of codanin-1 homolog in Drosophila we investigated the cellular localization and possible involvement of codanin-1 during the cell cycle.

Codanin-1, the protein encoded by the gene mutated in congenital dyserythropoietic anemia type I (CDAN1), is cell cycle-regulated.

Over the past decade, our understanding of intestinal immunology has been transformed by the realization that the gut microbiota shapes immune development, function and disease susceptibility. Strikingly, the enteric nervous system is now emerging as a cellular hub that sense microbiota-derived signals and rapidly orchestrate immunological outcomes. Here, I discuss recent advances in our understanding of intestinal neuroimmune communications, their modulation by the gut microbiota, and the consequent impact on host immunity, in health and disease.

Ménage à trois: regulation of host immunity by enteric neuro-immune-microbiota cross talks.

Short-chain fatty acids are processed from indigestible dietary fibers by gut bacteria and have immunomodulatory properties. Here, we investigate propionic acid (PA) in multiple sclerosis (MS), an autoimmune and neurodegenerative disease. Serum and feces of subjects with MS exhibited significantly reduced PA amounts compared with controls, particularly after the first relapse. In a proof-of-concept study, we supplemented PA to therapy-naive MS patients and as an add-on to MS immunotherapy. After 2 weeks of PA intake, we observed a significant and sustained increase of functionally competent regulatory T (Treg) cells, whereas Th1 and Th17 cells decreased significantly. Post-hoc analyses revealed a reduced annual relapse rate, disability stabilization, and reduced brain atrophy after 3 years of PA intake. Functional microbiome analysis revealed increased expression of Treg-cell-inducing genes in the intestine after PA intake. Furthermore, PA normalized Treg cell mitochondrial function and morphology in MS. Our findings suggest that PA can serve as a potent immunomodulatory supplement to MS drugs.

Propionic Acid Shapes the Multiple Sclerosis Disease Course by an Immunomodulatory Mechanism.

The protein translocated intimin receptor (Tir) from enteropathogenic  shares sequence similarity with the host cellular immunoreceptor tyrosine-based inhibition motifs (ITIMs). The ITIMs of Tir are required for Tir-mediated immune inhibition and evasion of host immune responses. However, the underlying molecular mechanism by which Tir regulates immune inhibition remains unclear. Here we demonstrated that β-arrestin 2, which is involved in the G-protein-coupled receptor (GPCR) signal pathway, interacted with Tir in an ITIM-dependent manner. For the molecular mechanism, we found that β-arrestin 2 enhanced the recruitment of SHP-1 to Tir. The recruited SHP-1 inhibited K63-linked ubiquitination of TRAF6 by dephosphorylating TRAF6 at Tyr288, and inhibited K63-linked ubiquitination and phosphorylation of TAK1 by dephosphorylating TAK1 at Tyr206, which cut off the downstream signal transduction and subsequent cytokine production. Moreover, the inhibitory effect of Tir on immune responses was diminished in β-arrestin 2-deficient mice and macrophages. These findings suggest that β-arrestin 2 is a key regulator in Tir-mediated immune evasion, which could serve as a new therapeutic target for bacterial infectious diseases.

β-arrestin 2 quenches TLR signaling to facilitate the immune evasion of EPEC.

The intestinal barrier protects intestinal cells from microbes and antigens in the lumen-breaches can alter the composition of the intestinal microbiota, the enteric immune system, and metabolism. We performed a screen to identify molecules that disrupt and support the intestinal epithelial barrier and tested their effects in mice.

High-Throughput Screen Identifies Host and Microbiota Regulators of Intestinal Barrier Function.

Celiac disease (CD) is a complex immune-mediated chronic disease characterized by a consistent inflammation of the gastrointestinal tract induced by gluten intake in genetically predisposed individuals. Although initiated by the interaction between digestion-derived gliadin, a gluten component, peptides, and the intestinal epithelium, the disorder is highly complex and involving other components of the intestine, such as the immune system. Therefore, conventional model systems, mainly based on two- or three-dimension cell cultures and co-cultures, cannot fully recapitulate such a complex disease. The development of mouse models has facilitated the study of different interacting cell types involved in the disorder, together with the impact of environmental factors. However, such in vivo models are often expensive and time consuming. Here we propose an organ ex vivo culture (gut-ex-vivo system) based on small intestines from gluten-sensitive mice cultivated in a dynamic condition, able to fully recapitulate the biochemical and morphological features of the mouse model exposed to gliadin (4 weeks), in 16 h. Indeed, upon gliadin exposure, we observed: i) a down-regulation of cystic fibrosis transmembrane regulator (CFTR) and an up-regulation of transglutaminase 2 (TG2) at both mRNA and protein levels; ii) increased intestinal permeability associated with deregulated tight junction protein expression; iii) induction and production of pro-inflammatory cytokines such as interleukin (IL)-15, IL-17 and interferon gamma (IFNγ); and iv) consistent alteration of intestinal epithelium/villi morphology. Altogether, these data indicate that the proposed model can be efficiently used to study the pathogenesis of CD, test new or repurposed molecules to accelerate the search for new treatments, and to study the impact of the microbiome and derived metabolites, in a time- and cost- effective manner.

Article	Year
Nek1 shares structural and functional similarities with NIMA kinase. Biochimica et biophysica acta\| PubMed ID: 16603261	2006
Nek7 kinase is enriched at the centrosome, and is required for proper spindle assembly and mitotic progression. FEBS letters\| PubMed ID: 17101132	2006
Codanin-1, the protein encoded by the gene mutated in congenital dyserythropoietic anemia type I (CDAN1), is cell cycle-regulated. Haematologica\| PubMed ID: 19336738	2009
Ménage à trois: regulation of host immunity by enteric neuro-immune-microbiota cross talks. Current opinion in neurobiology\| PubMed ID: 31809998	2020
Propionic Acid Shapes the Multiple Sclerosis Disease Course by an Immunomodulatory Mechanism. Cell\| PubMed ID: 32160527	2020
β-arrestin 2 quenches TLR signaling to facilitate the immune evasion of EPEC. Gut microbes\| PubMed ID: 32403971	2020
High-Throughput Screen Identifies Host and Microbiota Regulators of Intestinal Barrier Function. Gastroenterology\| PubMed ID: 32653496	2020
Gut-Ex-Vivo system as a model to study gluten response in celiac disease. Cell death discovery\| PubMed ID: 33712560	2021

Nissan Yissachar

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