Inflammation-Associated Microbiota Composition Across Domestic Animals

Domestic animals represent important resources for understanding shared mechanisms underlying complex natural diseases that arise due to both genetic and environmental factors. Intestinal inflammation, particularly inflammatory bowel disease (IBD), is a significant health challenge in humans and domestic animals. While the etiology of IBD is multifactorial, imbalance of symbiotic gut microbiota has been hypothesized to play a central role in disease pathophysiology. Advances in genomic sequencing and analytical pipelines have enabled researchers to decipher the composition of the intestinal microbiota during health and in the context of naturally occurring diseases. This review compiles microbiome genomic data across domestic species and highlights a common occurrence of gut microbiome dysbiosis during idiopathic intestinal inflammation in multiple species, including dogs, cats, horses, cows, and pigs. Current microbiome data obtained from animals with intestinal inflammation are mostly limited to taxonomical analyses in association with broad clinical phenotype. In general, a pathogen or pathosymbiont were not detected. Rather, functional potential of the altered microbiota has been suggested to be one of the key etiologic factors. Among the domestic species studied, canine analyses are currently the most advanced with incorporation of functional profiling of microbiota. Canine IBD parallels features of the disease in humans, thus canines represent a strong natural model for human IBD. While deeper analyses of metagenomic data, coupled with host molecular analyses are needed, comparative studies across domestic species can reveal shared microbial alterations and regulatory mechanisms that will improve our understanding of intestinal inflammation in both animals and humans

Microbiota Inhibit Epithelial Pathogen Adherence by Epigenetically Regulating C-Type Lectin Expression

Numerous bacterial pathogens infect the mammalian host by initially associating with epithelial cells that line the intestinal lumen. Recent work has revealed that commensal bacteria that reside in the intestine promote defense against pathogenic infection, however whether the microbiota direct host pathways that alter pathogen adherence is not well-understood. Here, by comparing germ-free mice, we identify that the microbiota decrease bacterial pathogen adherence and dampen epithelial expression of the cell surface glycoprotein C-type lectin 2e (Clec2e). Functional studies revealed that overexpression of this lectin promotes adherence of intestinal bacterial pathogens to mammalian cells. Interestingly, microbiota-sensitive downregulation of Clec2e corresponds with decreased histone acetylation of the Clec2e gene in intestinal epithelial cells. Histone deacetylation and transcriptional regulation of Clec2e depends on expression and recruitment of the histone deacetylase HDAC3. Thus, commensal bacteria epigenetically instruct epithelial cells to decrease expression of a C-type lectin that promotes pathogen adherence, revealing a novel mechanism for how the microbiota promote innate defense against infection

Mechano-Coupling and Regulation of Contractility by the Vinculin Tail Domain

Vinculin binds to multiple focal adhesion and cytoskeletal proteins and has been implicated in transmitting mechanical forces between the actin cytoskeleton and integrins or cadherins. It remains unclear to what extent the mechano-coupling function of vinculin also involves signaling mechanisms. We report the effect of vinculin and its head and tail domains on force transfer across cell adhesions and the generation of contractile forces. The creep modulus and the adhesion forces of F9 mouse embryonic carcinoma cells (wild-type), vinculin knock-out cells (vinculin −/−), and vinculin −/− cells expressing either the vinculin head domain, tail domain, or full-length vinculin (rescue) were measured using magnetic tweezers on fibronectin-coated super-paramagnetic beads. Forces of up to 10 nN were applied to the beads. Vinculin −/− cells and tail cells showed a slightly higher incidence of bead detachment at large forces. Compared to wild-type, cell stiffness was reduced in vinculin −/− and head cells and was restored in tail and rescue cells. In all cell lines, the cell stiffness increased by a factor of 1.3 for each doubling in force. The power-law exponent of the creep modulus was force-independent and did not differ between cell lines. Importantly, cell tractions due to contractile forces were suppressed markedly in vinculin −/− and head cells, whereas tail cells generated tractions similar to the wild-type and rescue cells. These data demonstrate that vinculin contributes to the mechanical stability under large external forces by regulating contractile stress generation. Furthermore, the regulatory function resides in the tail domain of vinculin containing the paxillin-binding site

Histone deacetylase 5 regulates the inflammatory response of macrophages

Modifying the chromatin structure and interacting with non-histone proteins, histone deacetylases (HDAC) are involved in vital cellular processes at different levels. We here specifically investigated the direct effects of HDAC5 in macrophage activation in response to bacterial or cytokine stimuli. Using murine and human macrophage cell lines, we studied the expression profile and the immunological function of HDAC5 at transcription and protein level in over-expression as well as RNA interference experiments. Toll-like receptor-mediated stimulation of murine RAW264.7 cells significantly reduced HDAC5 mRNA within 7 hrs but presented baseline levels after 24 hrs, a mechanism that was also found for Interferon-γ treatment. If treated with lipopolysaccharide, RAW264.7 cells transfected for over-expression only of full-length but not of mutant HDAC5, significantly elevated secretion of tumour necrosis factor α and of the monocyte chemotactic protein-1. These effects were accompanied by increased nuclear factor-κB activity. Accordingly, knock down of HDAC5-mRNA expression using specific siRNA significantly reduced the production of these cytokines in RAW264.7 or human U937 cells. Taken together, our results suggest a strong regulatory function of HDAC5 in the pro-inflammatory response of macrophages

La matriz extracelular: morfología, función y biotensegridad (parte I)

La matriz extracelular (MEC) representa una red tridimensional que engloba todos los órganos, tejidos y células del organismo. Constituye un filtro biofísico de protección, nutrición e inervación celular y el terreno para la respuesta inmune, angiogénesis, fibrosis y regeneración tisular. Y representa el medio de transmisión de fuerzas mecánicas a la membrana basal, que a través de las integrinas soporta el sistema de tensegridad y activa los mecanismos epigenéticos celulares. La alteración de la MEC supone la pérdida de su función de filtro eficaz, nutrición, eliminación, denervación celular, pérdida de la capacidad de regeneración y cicatrización y alteración de la transmisión mecánica o mecanotransducción. También la pérdida del sustrato para una correcta respuesta inmune ante agentes infecciosos, tumorales y tóxicos. Los tumores son tejidos funcionales conectados y dependientes del microambiente. El microambiente tumoral, constituido por la MEC, células del estroma y la propia respuesta inmune, son determinantes de la morfología y clasificación tumoral, agresividad clínica, pronóstico y respuesta al tratamiento del tumor. Tanto en condiciones fisiológicas como patológicas, la comunicación recíproca entre células del estroma y el parénquima dirige la expresión génica. La capacidad oncogénica del estroma procede tanto de los fibroblastos asociados al tumor como de la celularidad de la respuesta inmune y la alteración de la tensegridad por la MEC. La transición epitelio-mesenquimal es el cambio que transforma una célula normal o «benigna» en «maligna». El citoesqueleto pseudomesenquimal otorga las propiedades de migración, invasión y diseminación. Y viceversa, el fenotipo maligno es reversible a través de la corrección de las claves que facilita el microambiente tumoral.Extracellular matrix (ECM) is a three-dimensional network that envelopes all the organs, tissues and cells of the body. A biophysical filter that provides protection, nutrition and cell innervation, it is the site for immune response, angiogenesis, fibrosis and tissue regeneration. It is also the transport medium for mechanical forces to the basal membrane through integrins that support the tensegrity system, activating cellular epigenetic mechanisms. The disruption of the ECM leads to a functional loss of nutrition, elimination, cell innervation, regenerative capacity and wound healing as well as alterations in mechanical transduction. This loss also disrupts the immune response to pathogens, tumour cells and toxins. Tumours are functionally connected tissues which depend on the microenvironment. This tumour microenvironment, made up of ECM, stromal cells and the immune response, determines the morphology and tumour histopathological classification, clinical behaviour, prognosis and immune response to the tumour. Both in physiological and pathological conditions, reciprocity in the communication between stromal and parenchymal cells determine gene expression. The oncogenic capacity of the stroma depends on tumour associated fibroblasts, immune system cellularity and disruption of tensegrity by ECM. Epithelial-mesenchymal transition is the change that transforms a normal or benign cell into a malignant cell. The «pseudo-mensenchymal» cytoskeleton is responsible for migration, invasion and dissemination, and vice-versa, the malignant phenotype is reversible through the correction of the microenvironmental factors that favour tumour growth.Noguera Salva, Rosa, [email protected]

A microfluidic bioreactor with integrated transepithelial electrical resistance (TEER) measurement electrodes for evaluation of renal epithelial cells

We have developed a bilayer microfluidic system with integrated transepithelial electrical resistance (TEER) measurement electrodes to evaluate kidney epithelial cells under physiologically relevant fluid flow conditions. The bioreactor consists of apical and basolateral fluidic chambers connected via a transparent microporous membrane. The top chamber contains microfluidic channels to perfuse the apical surface of the cells. The bottom chamber acts as a reservoir for transport across the cell layer and provides support for the membrane. TEER electrodes were integrated into the device to monitor cell growth and evaluate cell–cell tight junction integrity. Immunofluorescence staining was performed within the microchannels for ZO-1 tight junction protein and acetylated Α-tubulin (primary cilia) using human renal epithelial cells (HREC) and MDCK cells. HREC were stained for cytoskeletal F-actin and exhibited disassembly of cytosolic F-actin stress fibers when exposed to shear stress. TEER was monitored over time under normal culture conditions and after disruption of the tight junctions using low Ca 2+ medium. The transport rate of a fluorescently labeled tracer molecule (FITC-inulin) was measured before and after Ca 2+ switch and a decrease in TEER corresponded with a large increase in paracellular inulin transport. This bioreactor design provides an instrumented platform with physiologically meaningful flow conditions to study various epithelial cell transport processes. Biotechnol. Bioeng. 2010;107:707–716. © 2010 Wiley Periodicals, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78065/1/22835_ftp.pd

Use of RNAlater in fluorescence-activated cell sorting (FACS) reduces the fluorescence from GFP but not from DsRed

<p>Abstract</p> <p>Background</p> <p>Flow cytometry utilizes signals from fluorescent markers to separate targeted cell populations for gene expression studies. However, the stress of the FACS process could change normal gene expression profiles. RNAlater could be used to stop such changes in original gene expression profiles through its ability to denature RNase and other proteins. The normal conformational structure of fluorescent proteins must be maintained in order to fluoresce. Whether or not RNAlater would affect signals from different types of intrinsic fluorescent proteins is crucial to its use in flow cytometry; this question has not been investigated in detail.</p> <p>Findings</p> <p>To address this question, we analyzed the effect of RNAlater on fluorescence intensity of GFP, YFP, DsRed and small fluorescent molecules attached to secondary antibodies (Cy2 and Texas-Red) when used in flow cytometry. FACS results were confirmed with fluorescence microscopy. Our results showed that exposure of YFP and GFP containing cells to RNAlater reduces the intensity of their fluorescence to such an extent that separation of such labeled cells is difficult if not impossible. In contrast, signals from DsRed2, Cy2 and Texas-Red were not affected by RNAlater treatment. In addition, the background fluorescence and clumping of dissociated cells are altered by RNAlater treatment.</p> <p>Conclusions</p> <p>When considering gene expression studies using cell sorting with RNAlater, DsRed is the fluorescent protein of choice while GFP/YFP have severe limitations because of their reduced fluorescence. It is necessary to examine the effects of RNAlater on signals from fluorescent markers and the physical properties (e.g., clumping) of the cells before considering its use in cell sorting.</p

Breakdown of the Endothelial Barrier Function in Tumor Cell Transmigration

The ability of tumor cells to metastasize is associated with a poor prognosis for cancer. During the process of metastasis, tumor cells circulating in the blood or lymph vessels can adhere to, and potentially transmigrate through, the endothelium and invade the connective tissue. We studied the effectiveness of the endothelium as a barrier against the invasion of 51 tumor cell lines into a three-dimensional collagen matrix. Only nine tumor cell lines showed attenuated invasion in the presence of an endothelial cell monolayer, whereas 17 cell lines became invasive or showed a significantly increased invasion. Endothelial cells cocultured with invasive tumor cells increased chemokine gene expression of IL-8 and Gro-β. Expression of the IL-8 and Gro-β receptor, CXCR2, was upregulated in invasive tumor cells. Addition of IL-8 or Gro-β increased tumor cell invasiveness by more than twofold. Tumor cell variants selected for high CXCR2 expression were fourfold more invasive in the presence of an endothelial cell layer, whereas CXCR2 siRNA knock-down cells were fivefold less invasive. We demonstrate that Gro-β and IL-8 secreted by endothelial cells, together with CXCR2 receptor expression on invasive tumor cells, contribute to the breakdown of the endothelial barrier by enhancing tumor cell force generation and cytoskeletal remodeling dynamics

Microarray analysis of gene expression profiles of cardiac myocytes and fibroblasts after mechanical stress, ionising or ultraviolet radiation

BACKGROUND: During excessive pressure or volume overload, cardiac cells are subjected to increased mechanical stress (MS). We set out to investigate how the stress response of cardiac cells to MS can be compared to genotoxic stresses induced by DNA damaging agents. We chose for this purpose to use ionising radiation (IR), which during mediastinal radiotherapy can result in cardiac tissue remodelling and diminished heart function, and ultraviolet radiation (UV) that in contrast to IR induces high concentrations of DNA replication- and transcription-blocking lesions. RESULTS: Cultures enriched for neonatal rat cardiac myocytes (CM) or fibroblasts were subjected to any one of the three stressors. Affymetrix microarrays, analysed with Linear Modelling on Probe Level, were used to determine gene expression patterns at 24 hours after (the start of) treatment. The numbers of differentially expressed genes after UV were considerably higher than after IR or MS. Remarkably, after all three stressors the predominant gene expression response in CM-enriched fractions was up-regulation, while in fibroblasts genes were more frequently down-regulated. To investigate the activation or repression of specific cellular pathways, genes present on the array were assigned to 25 groups, based on their biological function. As an example, in the group of cholesterol biosynthesis a significant proportion of genes was up-regulated in CM-enriched fractions after MS, but down-regulated after IR or UV. CONCLUSION: Gene expression responses after the types of cellular stress investigated (MS, IR or UV) have a high stressor and cell type specificity

Cell type-specific mechanisms coupling protease-activated receptor-1 to infectious colitis pathogenesis

Background: Protease-activated receptor-1 (PAR-1) plays a major role in multiple disease processes, including colitis. Understanding the mechanisms coupling PAR-1 to disease pathogenesis is complicated by the fact that PAR-1 is broadly expressed across multiple cell types. Objective: Determine the specific contributions of PAR-1 expressed by macrophages and colonic enterocytes to infectious colitis. Methods: Mice carrying a conditional PAR-1 allele were generated and bred to mice expressing Cre recombinase in a myeloid- (PAR-1ΔM) or enterocyte-specific (PAR-1ΔEPI) fashion. Citrobacter rodentium colitis pathogenesis was analyzed in mice with global PAR-1 deletion (PAR-1−/−) and cell type-specific deletions. Results: Constitutive deletion of PAR-1 had no significant impact on weight loss, crypt hypertrophy, crypt abscess formation, or leukocyte infiltration in Citrobacter colitis. However, colonic shortening was significantly blunted in infected PAR-1−/− mice, and these animals exhibited decreased local levels of IL-1β, IL-22, IL-6, and IL-17A. In contrast, infected PAR-1ΔM mice lost less weight and had fewer crypt abscesses relative to controls. PAR-1ΔM mice had diminished CD3+ T cell infiltration into colonic tissue, but macrophage and CD4+ T cell infiltration were similar to controls. Also contrasting results in global knockouts, PAR-1ΔM mice exhibited lower levels of IL-1β, but not Th17-related cytokines (ie, IL-22, IL-6, IL-17A). Infected PAR-1ΔEPI mice exhibited increased crypt hypertrophy and crypt abscess formation, but local cytokine elaboration was similar to controls. Conclusions: These studies reveal complex, cell type-specific roles for PAR-1 in modulating the immune response to Citrobacter colitis that are not readily apparent in analyses limited to mice with global PAR-1 deficiency