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

Understanding Sensory Nerve Mechanotransduction through Localized Elastomeric Matrix Control

BACKGROUND: While neural systems are known to respond to chemical and electrical stimulation, the effect of mechanics on these highly sensitive cells is still not well understood. The ability to examine the effects of mechanics on these cells is limited by existing approaches, although their overall response is intimately tied to cell-matrix interactions. Here, we offer a novel method, which we used to investigate stretch-activated mechanotransduction on nerve terminals of sensory neurons through an elastomeric interface. METHODOLOGY/PRINCIPAL FINDINGS: To apply mechanical force on neurites, we cultured dorsal root ganglion neurons on an elastic substrate, polydimethylsiloxane (PDMS), coated with extracellular matrices (ECM). We then implemented a controlled indentation scheme using a glass pipette to mechanically stimulate individual neurites that were adjacent to the pipette. We used whole-cell patch clamping to record the stretch-activated action potentials on the soma of the single neurites to determine the mechanotransduction-based response. When we imposed specific mechanical force through the ECM, we noted a significant neuronal action potential response. Furthermore, because the mechanotransduction cascade is known to be directly affected by the cytoskeleton, we investigated the cell structure and its effects. When we disrupted microtubules and actin filaments with nocodozale or cytochalasin-D, respectively, the mechanically induced action potential was abrogated. In contrast, when using blockers of channels such as TRP, ASIC, and stretch-activated channels while mechanically stimulating the cells, we observed almost no change in action potential signalling when compared with mechanical activation of unmodified cells. CONCLUSIONS/SIGNIFICANCE: These results suggest that sensory nerve terminals have a specific mechanosensitive response that is related to cell architecture

Fasting and High-Fat Diet Alter Histone Deacetylase Expression in the Medial Hypothalamus

Increasing attention is now being given to the epigenetic regulation of animal and human behaviors including the stress response and drug addiction. Epigenetic factors also influence feeding behavior and metabolic phenotypes, such as obesity and insulin sensitivity. In response to fasting and high-fat diets, the medial hypothalamus changes the expression of neuropeptides regulating feeding, metabolism, and reproductive behaviors. Histone deacetylases (HDACs) are involved in the epigenetic control of gene expression and alter behavior in response to a variety of environmental factors. Here, we examined the expression of HDAC family members in the medial hypothalamus of mice in response to either fasting or a high-fat diet. In response to fasting, HDAC3 and −4 expression levels increased while HDAC10 and −11 levels decreased. Four weeks on a high-fat diet resulted in the increased expression of HDAC5 and −8. Moreover, fasting decreased the number of acetylated histone H3- and acetylated histone H4-positive cells in the ventrolateral subdivision of the ventromedial hypothalamus. Therefore, HDACs may be implicated in altered gene expression profiles in the medial hypothalamus under different metabolic states

Histone Deacetylase 3 Depletion in Osteo/Chondroprogenitor Cells Decreases Bone Density and Increases Marrow Fat

Histone deacetylase (Hdac)3 is a nuclear enzyme that contributes to epigenetic programming and is required for embryonic development. To determine the role of Hdac3 in bone formation, we crossed mice harboring loxP sites around exon 7 of Hdac3 with mice expressing Cre recombinase under the control of the osterix promoter. The resulting Hdac3 conditional knockout (CKO) mice were runted and had severe deficits in intramembranous and endochondral bone formation. Calvarial bones were significantly thinner and trabecular bone volume in the distal femur was decreased 75% in the Hdac3 CKO mice due to a substantial reduction in trabecular number. Hdac3-CKO mice had fewer osteoblasts and more bone marrow adipocytes as a proportion of tissue area than their wildtype or heterozygous littermates. Bone formation rates were depressed in both the cortical and trabecular regions of Hdac3 CKO femurs. Microarray analyses revealed that numerous developmental signaling pathways were affected by Hdac3-deficiency. Thus, Hdac3 depletion in osterix-expressing progenitor cells interferes with bone formation and promotes bone marrow adipocyte differentiation. These results demonstrate that Hdac3 inhibition is detrimental to skeletal health

Macrophage phenotype is associated with disease severity in preterm infants with chronic lung disease.

The etiology of persistent lung inflammation in preterm infants with chronic lung disease of prematurity (CLD) is poorly characterized, hampering efforts to stratify prognosis and treatment. Airway macrophages are important innate immune cells with roles in both the induction and resolution of tissue inflammation

IL-10–producing Tfh cells accumulate with age and link inflammation with age-related immune suppression

Aging results in profound immune dysfunction, resulting in the decline of vaccine responsiveness previously attributed to irreversible defects in the immune system. In addition to increased interleukin-6 (IL-6), we found aged mice exhibit increased systemic IL-10 that requires forkhead box P3–negative (FoxP3−), but not FoxP3+, CD4+T cells. Most IL-10–producing cells manifested a T follicular helper (Tfh) phenotype and required the Tfh cytokines IL-6 and IL-21 for their accrual, so we refer to them as Tfh10 cells. IL-21 was also required to maintain normal serum levels of IL-6 and IL-10. Notably, antigen-specific Tfh10 cells arose after immunization of aged mice, and neutralization of IL-10 receptor signaling significantly restored Tfh-dependent antibody responses, whereas depletion of FoxP3+ regulatory and follicular regulatory cells did not. Thus, these data demonstrate that immune suppression with age is reversible and implicate Tfh10 cells as an intriguing link between “inflammaging” and impaired immune responses with age

Importance of Non-Selective Cation Channel TRPV4 Interaction with Cytoskeleton and Their Reciprocal Regulations in Cultured Cells

BACKGROUND: TRPV4 and the cellular cytoskeleton have each been reported to influence cellular mechanosensitive processes as well as the development of mechanical hyperalgesia. If and how TRPV4 interacts with the microtubule and actin cytoskeleton at a molecular and functional level is not known. METHODOLOGY AND PRINCIPAL FINDINGS: We investigated the interaction of TRPV4 with cytoskeletal components biochemically, cell biologically by observing morphological changes of DRG-neurons and DRG-neuron-derived F-11 cells, as well as functionally with calcium imaging. We find that TRPV4 physically interacts with tubulin, actin and neurofilament proteins as well as the nociceptive molecules PKCepsilon and CamKII. The C-terminus of TRPV4 is sufficient for the direct interaction with tubulin and actin, both with their soluble and their polymeric forms. Actin and tubulin compete for binding. The interaction with TRPV4 stabilizes microtubules even under depolymerizing conditions in vitro. Accordingly, in cellular systems TRPV4 colocalizes with actin and microtubules enriched structures at submembranous regions. Both expression and activation of TRPV4 induces striking morphological changes affecting lamellipodial, filopodial, growth cone, and neurite structures in non-neuronal cells, in DRG-neuron derived F11 cells, and also in IB4-positive DRG neurons. The functional interaction of TRPV4 and the cytoskeleton is mutual as Taxol, a microtubule stabilizer, reduces the Ca2+-influx via TRPV4. CONCLUSIONS AND SIGNIFICANCE: TRPV4 acts as a regulator for both, the microtubule and the actin. In turn, we describe that microtubule dynamics are an important regulator of TRPV4 activity. TRPV4 forms a supra-molecular complex containing cytoskeletal proteins and regulatory kinases. Thereby it can integrate signaling of various intracellular second messengers and signaling cascades, as well as cytoskeletal dynamics. This study points out the existence of cross-talks between non-selective cation channels and cytoskeleton at multiple levels. These cross talks may help us to understand the molecular basis of the Taxol-induced neuropathic pain development commonly observed in cancer patients

Magnetic Forces And Magnetized Biomaterials Provide Dynamic Flux Information During Bone Regeneration

The fascinating prospect to direct tissue regeneration by magnetic activation has been recently explored. In this study we investigate the possibility to boost bone regeneration in an experimental defect in rabbit femoral condyle by combining static magnetic fields and magnetic biomaterials. NdFeB permanent magnets are implanted close to biomimetic collagen/hydroxyapatite resorbable scaffolds magnetized according to two different protocols. Permanent magnet only or non-magnetic scaffolds are used as controls. Bone tissue regeneration is evaluated at 12 weeks from surgery from a histological, histomorphometric and biomechanical point of view. The reorganization of the magnetized collagen fibers under the effect of the static magnetic field generated by the permanent magnet produces a highly-peculiar bone pattern, with highly-interconnected trabeculae orthogonally oriented with respect to the magnetic field lines. In contrast, only partial defect healing is achieved within the control groups. We ascribe the peculiar bone regeneration to the transfer of micro-environmental information, mediated by collagen fibrils magnetized by magnetic nanoparticles, under the effect of the static magnetic field. These results open new perspectives on the possibility to improve implant fixation and control the morphology and maturity of regenerated bone providing “in site” forces by synergically combining static magnetic fields and biomaterials