Involvement of RhoA-mediated Ca(2+ )sensitization in antigen-induced bronchial smooth muscle hyperresponsiveness in mice

BACKGROUND: It has recently been suggested that RhoA plays an important role in the enhancement of the Ca(2+ )sensitization of smooth muscle contraction. In the present study, a participation of RhoA-mediated Ca(2+ )sensitization in the augmented bronchial smooth muscle (BSM) contraction in a murine model of allergic asthma was examined. METHODS: Ovalbumin (OA)-sensitized BALB/c mice were repeatedly challenged with aerosolized OA and sacrificed 24 hours after the last antigen challenge. The contractility and RhoA protein expression of BSMs were measured by organ-bath technique and immunoblotting, respectively. RESULTS: Repeated OA challenge to sensitized mice caused a BSM hyperresponsiveness to acetylcholine (ACh), but not to high K(+)-depolarization. In α-toxin-permeabilized BSMs, ACh induced a Ca(2+ )sensitization of contraction, which is sensitive to Clostridium botulinum C3 exoenzyme, indicating that RhoA is implicated in this Ca(2+ )sensitization. Interestingly, the ACh-induced, RhoA-mediated Ca(2+ )sensitization was significantly augmented in permeabilized BSMs of OA-challenged mice. Moreover, protein expression of RhoA was significantly increased in the hyperresponsive BSMs. CONCLUSION: These findings suggest that the augmentation of Ca(2+ )sensitizing effect, probably via an up-regulation of RhoA protein, might be involved in the enhanced BSM contraction in antigen-induced airway hyperresponsiveness

A Commensal Helicobacter sp. of the Rodent Intestinal Flora Activates TLR2 and NOD1 Responses in Epithelial Cells

Helicobacter spp. represent a proportionately small but significant component of the normal intestinal microflora of animal hosts. Several of these intestinal Helicobacter spp. are known to induce colitis in mouse models, yet the mechanisms by which these bacteria induce intestinal inflammation are poorly understood. To address this question, we performed in vitro co-culture experiments with mouse and human epithelial cell lines stimulated with a selection of Helicobacter spp., including known pathogenic species as well as ones for which the pathogenic potential is less clear. Strikingly, a member of the normal microflora of rodents, Helicobacter muridarum, was found to be a particularly strong inducer of CXC chemokine (Cxcl1/KC, Cxcl2/MIP-2) responses in a murine intestinal epithelial cell line. Time-course studies revealed a biphasic pattern of chemokine responses in these cells, with H. muridarum lipopolysaccharide (LPS) mediating early (24–48 h) responses and live bacteria seeming to provoke later (48–72 h) responses. H. muridarum LPS per se was shown to induce CXC chemokine production in HEK293 cells stably expressing Toll-like receptor 2 (TLR2), but not in those expressing TLR4. In contrast, live H. muridarum bacteria were able to induce NF-κB reporter activity and CXC chemokine responses in TLR2–deficient HEK293 and in AGS epithelial cells. These responses were attenuated by transient transfection with a dominant negative construct to NOD1, and by stable expression of NOD1 siRNA, respectively. Thus, the data suggest that both TLR2 and NOD1 may be involved in innate immune sensing of H. muridarum by epithelial cells. This work identifies H. muridarum as a commensal bacterium with pathogenic potential and underscores the potential roles of ill-defined members of the normal flora in the initiation of inflammation in animal hosts. We suggest that H. muridarum may act as a confounding factor in colitis model studies in rodents

Luminal-Applied Flagellin Is Internalized by Polarized Intestinal Epithelial Cells and Elicits Immune Responses via the TLR5 Dependent Mechanism

Bacteria release flagellin that elicits innate responses via Toll-like receptor 5 (TLR5). Here, we investigated the fate of apically administrated full length flagellin from virulent and avirulent bacteria, along with truncated recombinant flagellin proteins in intestinal epithelial cells and cellular responses. Flagellin was internalized by intestinal epithelial cell (IEC) monolayers of IEC-18. Additionally, apically applied flagellin was internalized by polarized human Caco-2BBe and T-84 cells in a TLR5 dependent mechanism. More, flagellin exposure did not affect the integrity of intestinal monolayers. With immunofluorescent staining, internalized flagellin was detected in both early endosomes as well as lysosomes. We found that apical exposure of polarized Caco-2BBe and T-84 to flagellin from purified Salmonella, Escherichia coli O83:H1 (isolate from Crohn’s lesion) or avirulent E. coli K12 induced comparable levels of basolateral IL-8 secretion. A recombinant protein representing the conserved amino (N) and carboxyl (C) domains (D) of the flagellin protein (ND1/2ECHCD2/1) induced IL-8 secretion from IEC similar to levels elicited by full-length flagellins. However, a recombinant flagellin protein containing only the D3 hypervariable region elicited no IL-8 secretion in both cell lines compared to un-stimulated controls. Silencing or blocking TLR5 in Caco-2BBe cells resulted in a lack of flagellin internalization and decreased IL-8 secretion. Furthermore, apical exposure to flagellin stimulated transepithelial migration of neutrophils and dendritic cells. The novel findings in this study show that luminal-applied flagellin is internalized by normal IEC via TLR5 and co-localizes to endosomal and lysosomal compartments where it is likely degraded as flagellin was not detected on the basolateral side of IEC cultures

Cardiac fibrosis in aging mice

Dystrophic cardiac calcinosis (DCC), also called epicardial and myocardial fibrosis and mineralization, has been detected in mice of a number of laboratory inbred strains, most commonly C3H/HeJ and DBA/2J. In previous mouse breeding studies between these DCC susceptible and the DCC-resistant strain C57BL/6J, 4 genetic loci harboring genes involved in DCC inheritance were identified and subsequently termed Dyscalc loci 1 through 4. Here, we report susceptibility to cardiac fibrosis, a sub-phenotype of DCC, at 12 and 20 months of age and close to natural death in a survey of 28 inbred mouse strains. Eight strains showed cardiac fibrosis with highest frequency and severity in the moribund mice. Using genotype and phenotype information of the 28 investigated strains, we performed genome-wide association studies (GWAS) and identified the most significant associations on chromosome (Chr) 15 at 72 million base pairs (Mb) (P < 10(-13)) and Chr 4 at 122 Mb (P < 10(-11)) and 134 Mb (P < 10(-7)). At the Chr 15 locus, Col22a1 and Kcnk9 were identified. Both have been reported to be morphologically and functionally important in the heart muscle. The strongest Chr 4 associations were located approximately 6 Mb away from the Dyscalc 2 quantitative trait locus peak within the boundaries of the Extl1 gene and in close proximity to the Trim63 and Cap1 genes. In addition, a single-nucleotide polymorphism association was found on chromosome 11. This study provides evidence for more than the previously reported 4 genetic loci determining cardiac fibrosis and DCC. The study also highlights the power of GWAS in the mouse for dissecting complex genetic traits.The authors thank Jesse Hammer and Josiah Raddar for technical assistance. Research reported in this publication was supported by the Ellison Medical Foundation, Parker B. Francis Foundation, and the National Institutes of Health (R01AR055225 and K01AR064766). Mouse colonies were supported by the National Institutes of Health under Award Number AG025707 for the Jackson Aging Center. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The Jackson Laboratory Shared Scientific Services were supported in part by a Basic Cancer Center Core Grant from the National Cancer Institute (CA34196).This is the author accepted manuscript. The final version is available from Springer via http://dx.doi.org/10.1007/s00335-016-9634-

'Gut health': a new objective in medicine?

'Gut health' is a term increasingly used in the medical literature and by the food industry. It covers multiple positive aspects of the gastrointestinal (GI) tract, such as the effective digestion and absorption of food, the absence of GI illness, normal and stable intestinal microbiota, effective immune status and a state of well-being. From a scientific point of view, however, it is still extremely unclear exactly what gut health is, how it can be defined and how it can be measured. The GI barrier adjacent to the GI microbiota appears to be the key to understanding the complex mechanisms that maintain gut health. Any impairment of the GI barrier can increase the risk of developing infectious, inflammatory and functional GI diseases, as well as extraintestinal diseases such as immune-mediated and metabolic disorders. Less clear, however, is whether GI discomfort in general can also be related to GI barrier functions. In any case, methods of assessing, improving and maintaining gut health-related GI functions are of major interest in preventive medicine

Gut-central nervous system axis is a target for nutritional therapies

Historically, in the 1950s, the chemist Linus Pauling established a relationship between decreased longevity and obesity. At this time, with the advent of studies involving the mechanisms that modulate appetite control, some researchers observed that the hypothalamus is the "appetite centre" and that peripheral tissues have important roles in the modulation of gut inflammatory processes and levels of hormones that control food intake. Likewise, the advances of physiological and molecular mechanisms for patients with obesity, type 2 diabetes mellitus, inflammatory bowel diseases, bariatric surgery and anorexia-associated diseases has been greatly appreciated by nutritionists. Therefore, this review highlights the relationship between the gut-central nervous system axis and targets for nutritional therapies

International Society of Sports Nutrition Position Stand: Probiotics.

Position statement: The International Society of Sports Nutrition (ISSN) provides an objective and critical review of the mechanisms and use of probiotic supplementation to optimize the health, performance, and recovery of athletes. Based on the current available literature, the conclusions of the ISSN are as follows: 1)Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host (FAO/WHO).2)Probiotic administration has been linked to a multitude of health benefits, with gut and immune health being the most researched applications.3)Despite the existence of shared, core mechanisms for probiotic function, health benefits of probiotics are strain- and dose-dependent.4)Athletes have varying gut microbiota compositions that appear to reflect the activity level of the host in comparison to sedentary people, with the differences linked primarily to the volume of exercise and amount of protein consumption. Whether differences in gut microbiota composition affect probiotic efficacy is unknown.5)The main function of the gut is to digest food and absorb nutrients. In athletic populations, certain probiotics strains can increase absorption of key nutrients such as amino acids from protein, and affect the pharmacology and physiological properties of multiple food components.6)Immune depression in athletes worsens with excessive training load, psychological stress, disturbed sleep, and environmental extremes, all of which can contribute to an increased risk of respiratory tract infections. In certain situations, including exposure to crowds, foreign travel and poor hygiene at home, and training or competition venues, athletes' exposure to pathogens may be elevated leading to increased rates of infections. Approximately 70% of the immune system is located in the gut and probiotic supplementation has been shown to promote a healthy immune response. In an athletic population, specific probiotic strains can reduce the number of episodes, severity and duration of upper respiratory tract infections.7)Intense, prolonged exercise, especially in the heat, has been shown to increase gut permeability which potentially can result in systemic toxemia. Specific probiotic strains can improve the integrity of the gut-barrier function in athletes.8)Administration of selected anti-inflammatory probiotic strains have been linked to improved recovery from muscle-damaging exercise.9)The minimal effective dose and method of administration (potency per serving, single vs. split dose, delivery form) of a specific probiotic strain depends on validation studies for this particular strain. Products that contain probiotics must include the genus, species, and strain of each live microorganism on its label as well as the total estimated quantity of each probiotic strain at the end of the product's shelf life, as measured by colony forming units (CFU) or live cells.10)Preclinical and early human research has shown potential probiotic benefits relevant to an athletic population that include improved body composition and lean body mass, normalizing age-related declines in testosterone levels, reductions in cortisol levels indicating improved responses to a physical or mental stressor, reduction of exercise-induced lactate, and increased neurotransmitter synthesis, cognition and mood. However, these potential benefits require validation in more rigorous human studies and in an athletic population