Gut bacterial composition in a mouse model of Parkinson's disease.

The mechanism of neurodegeneration in Parkinson's disease (PD) remains unknown but it has been hypothesised that the intestinal tract could be an initiating and contributing factor to the neurodegenerative processes. In PD patients as well as in animal models for PD, alpha-synuclein-positive enteric neurons in the colon and evidence of colonic inflammation have been demonstrated. Moreover, several studies reported pro-inflammatory bacterial dysbiosis in PD patients. Here, we report for the first time significant changes in the composition of caecum mucosal associated and luminal microbiota and the associated metabolic pathways in a rotenone-induced mouse model for PD. The mouse model for PD, induced by the pesticide rotenone, is associated with an imbalance in the gut microbiota, characterised by a significant decrease in the relative abundance of the beneficial commensal bacteria genus Bifidobacterium. Overall, intestinal bacterial dysbiosis might play an important role in both the disruption of intestinal epithelial integrity and intestinal inflammation, which could lead or contribute to the observed alpha-synuclein aggregation and PD pathology in the intestine and central nervous system in the oral rotenone mouse model of PD.

Circadian Rhythm and the Gut Microbiome.

Circadian rhythms are 24-h patterns regulating behavior, organs, and cells in living organisms. These rhythms align biological functions with regular and predictable environmental patterns to optimize function and health. Disruption of these rhythms can be detrimental resulting in metabolic syndrome, cancer, or cardiovascular disease, just to name a few. It is now becoming clear that the intestinal microbiome is also regulated by circadian rhythms via intrinsic circadian clocks as well as via the host organism. Microbiota rhythms are regulated by diet and time of feeding which can alter both microbial community structure and metabolic activity which can significantly impact host immune and metabolic function. In this review, we will cover how host circadian rhythms are generated and maintained, how host circadian rhythms can be disrupted, as well as the consequences of circadian rhythm disruption. We will further highlight the newly emerging literature indicating the importance of circadian rhythms of the intestinal microbiota.

NF-kappaB activation as a key mechanism in ethanol-induced disruption of the F-actin cytoskeleton and monolayer barrier integrity in intestinal epithelium.

Intestinal barrier disruption has been implicated in several intestinal and systemic disorders including alcoholic liver disease (ALD). Using monolayers of intestinal (Caco-2) cells, we showed that ethanol (EtOH) disrupts the barrier integrity via destabilization of the cytoskeleton. Because proinflammatory conditions are associated with activation of NF-kappa B (NF-kappaB), we hypothesized that EtOH induces disruption of cytoskeletal assembly and barrier integrity by activating NF-kappaB. Parental cells were pretreated with pharmacological modulators of NF-kappaB. Other cells were stably transfected with a dominant negative mutant for the NF-kappaB inhibitor, I-kappaBalpha. Monolayers of each cell type were exposed to EtOH and we then monitored monolayer barrier integrity (permeability); cytoskeletal stability and molecular dynamics (confocal microscopy and immunoblotting); intracellular levels of the I-kappaBalpha (immunoblotting); subcellular distribution and activity of NF-kappaB (immunoblotting and sensitive ELISA); and intracellular alterations in the 43kDa protein of the actin cytoskeleton, polymerized F-actin, and monomeric G-actin (SDS-PAGE fractionation). EtOH caused destabilizing alterations, including I-kappaBalpha degradation, NF-kappaB nuclear translocation, NF-kappaB subunit (p50 and p65) activation, actin disassembly (upward arrow G-, downward arrow F-), actin cytoskeleton instability, and barrier disruption. Inhibitors of NF-kappaB and stabilizers of I-kappaBalpha (e.g., MG-132, lactacystin, etc) prevented NF-kappaB activation while protecting against EtOH-induced injury. In transfected I-kappaBalpha mutant clones, stabilization of I-kappaBalpha to inactivate NF-kappaB protected against all measures of EtOH-induced injury. Our data support several novel mechanisms where NF-kappaB can affect the molecular dynamics of the F-actin cytoskeleton and intestinal barrier integrity under conditions of EtOH injury. (1) EtOH induces disruption of the F-actin cytoskeleton and of intestinal barrier integrity, in part, through I-kappaBalpha degradation and NF-kappaB activation; (2) The mechanism underlying this pathophysiological effect of the NF-kappaB appears to involve instability of the assembly of the subunit components of actin network.

Regulation of oxidant-induced intestinal permeability by metalloprotease-dependent epidermal growth factor receptor signaling.

Inflammatory bowel disease (IBD) affects more than 1 million Americans with more than 30,000 new cases diagnosed each year. IBD increases patient morbidity and susceptibility to colorectal cancer, yet its etiology remains unknown. Current models identify two key determinants of IBD pathogenesis: hyperpermeability of the gut epithelial barrier to bacterial products and an abnormal immune response to these products. Two factors seem critical for hyperpermeability: oxidant-induced stress and proinflammatory cytokines (e.g., tumor necrosis factor-alpha). The aim of this study was to investigate the role of oxidant stress-mediated transactivation of the epidermal growth factor receptor (EGFR) in intestinal hyperpermeability. This study used the Caco-2 human colonic epithelial cell in vitro model of intestinal epithelium. Cells were grown on inserts for permeability and signaling studies and glass coverslips for microscopy studies. show that oxidant-induced intestinal hyperpermeability can be blocked by specific inhibitors of the EGFR, tumor necrosis factor convertase (TACE) metalloprotease, transforming growth factor (TGF)-alpha, and mitogen-activated protein kinases, especially extracellular signal-regulated kinase 1/2. We also show that oxidant initiates these signaling events, in part by causing translocation of TACE to cell-cell contact zones. In this study, our data identify a novel mechanism for oxidant-induced intestinal hyperpermeability relevant to IBD. We propose a new intestinal permeability model in which oxidant transactivates EGFR signaling by activation of TACE and cleavage of precursor TGF-alpha. These data could have a significant effect on our view of IBD pathogenesis and provide new therapeutic targets for IBD treatment.

theta Isoform of protein kinase C alters barrier function in intestinal epithelium through modulation of distinct claudin isotypes: a novel mechanism for regulation of permeability.

Using monolayers of intestinal Caco-2 cells, we discovered that the isoform of protein kinase C (PKC), a member of the "novel" subfamily of PKC isoforms, is required for monolayer barrier function. However, the mechanisms underlying this novel effect remain largely unknown. Here, we sought to determine whether the mechanism by which PKC- disrupts monolayer permeability and dynamics in intestinal epithelium involves PKC--induced alterations in claudin isotypes. We used cell clones that we recently developed, clones that were transfected with varying levels of plasmid to either stably suppress endogenous PKC- activity (antisense, dominant-negative constructs) or to ectopically express PKC- activity (sense constructs). We then determined barrier function, claudin isotype integrity, PKC- subcellular activity, claudin isotype subcellular pools, and claudin phosphorylation. Antisense transfection to underexpress the PKC- led to monolayer instability as shown by reduced 1) endogenous PKC- activity, 2) claudin isotypes in the membrane and cytoskeletal pools ( downward arrowclaud-1, downward arrowclaud-4 assembly), 3) claudin isotype phosphorylation ( downward arrow phospho-serine, downward arrow phospho-threonine), 4) architectural stability of the claudin-1 and claudin-4 rings, and 5) monolayer barrier function. In these antisense clones, PKC- activity was also substantially reduced in the membrane and cytoskeletal cell fractions. In wild-type (WT) cells, PKC- (82 kDa) was both constitutively active and coassociated with claudin-1 (22 kDa) and claudin-4 (25 kDa), forming endogenous PKC-/claudin complexes. In a second series of studies, dominant-negative inhibition of the endogenous PKC- caused similar destabilizing effects on monolayer barrier dynamics, including claudin-1 and -4 hypophosphorylation, disassembly, and architectural instability as well as monolayer disruption. In a third series of studies, sense overexpression of the PKC- caused not only a mostly cytosolic distribution of this isoform (i.e., <12% in the membrane + cytoskeletal fractions, indicating PKC- inactivity) but also led to disruption of claudin assembly and barrier function of the monolayer. The conclusions of this study are that PKC- activity is required for normal claudin assembly and the integrity of the intestinal epithelial barrier. These effects of PKC- are mediated at the molecular level by changes in phosphorylation, membrane assembly, and/or organization of the subunit components of two barrier function proteins: claudin-1 and claudin-4 isotypes. The ability of PKC- to alter the dynamics of permeability protein claudins is a new function not previously ascribed to the novel subfamily of PKC isoforms.

Critical role of the atypical {lambda} isoform of protein kinase C (PKC-{lambda}) in oxidant-induced disruption of the microtubule cytoskeleton and barrier function of intestinal epithelium.

Oxidant injury to epithelial cells and gut barrier disruption are key factors in the pathogenesis of inflammatory bowel disease. Studying monolayers of intestinal (Caco-2) cells, we reported that oxidants disrupt the cytoskeleton and cause barrier dysfunction (hyperpermeability). Because the lambda isoform of protein kinase C (PKC-lambda), an atypical diacylglycerol-independent isozyme, is abundant in parental (wild type) Caco-2 cells and is translocated to the particulate fractions upon oxidant exposure, we hypothesized that PKC-lambda is critical to oxidative injury to the assembly and architecture of cytoskeleton and the intestinal barrier function. To this end, Caco-2 cells were transfected with an inducible plasmid, a tetracycline-responsive system, to create novel clones stably overexpressing native PKC-lambda. Other cells were transfected with a dominant-negative plasmid to stably inhibit the activity of native PKC-lambda. Cells were exposed to oxidant (H(2)O(2)) +/- modulators. Parental Caco-2 cells were treated similarly. We then monitored barrier function (fluorescein sulfonic acid clearance), microtubule cytoskeletal stability (confocal microscopy, immunoblotting), subcellular distribution of PKC-lambda (immunofluorescence, immunoblotting, immunoprecipitation), and PKC-lambda isoform activity (in vitro kinase assay). Monolayers were also processed to assess alterations in tubulin assembly, polymerized tubulin (S2, an index of cytoskeletal integrity), and monomeric tubulin (S1, an index of cytoskeletal disassembly) (polyacrylamide gel electrophoresis fractionation and immunoblotting. In parental cells, oxidant caused: 1) translocation of PKC-lambda from the cytosol to the particulate (membrane + cytoskeletal) fractions, 2) activation of native PKC-lambda, 3) tubulin pool instability (increased monomeric S1 and decreased polymerized S2), 4) disruption of cytoskeletal architecture, and 5) barrier dysfunction (hyperpermeability). In transfected clones, overexpression of the atypical (74 kDa) PKC-lambda isoform by itself ( approximately 3.2-fold increase) led to oxidant-like disruptive effects, including cytoskeletal and barrier hyperpermeability. Overexpressed PKC-lambda was mostly found in particulate cell fractions (with a smaller cytosolic distribution) indicating its activation. Disruption by PKC-lambda overexpression was also potentiated by oxidant challenge. Stable inactivation of endogenous PKC-lambda ( approximately 99.6%) by a dominant-negative protected against all measures of oxidant-induced disruption. We conclude that: 1) oxidant induces disruption of epithelial barrier integrity by disassembling the cytoskeleton, in large part, through the activation of PKC-lambda isoform; and 2) activation of PKC-lambda by itself appears to be sufficient for disruption of cellular cytoskeleton and monolayer barrier permeability. The unique ability to mediate an oxidant-like injury and cytoskeletal depolymerization and instability is a novel mechanism not previously attributed to the atypical subfamily of PKC isoforms.

Integrin alpha(M)beta(2)-mediated cell migration to fibrinogen and its recognition peptides.

Leukocyte migration is the hallmark of inflammation, and integrin alpha(M)beta(2) and its ligand fibrinogen (Fg) are key participants in this cellular response. Cells expressing wild-type or mutant alpha(M)beta(2) and Fg or its derivatives have been used to dissect the molecular requirements for this receptor-ligand pair to mediate cell migration. The major conclusions are that (a) Fg, its D fragment, and its P1 and P2 alpha(M)beta(2) recognition peptides support a chemotactic response; (b) when the I domain of alpha(L) was replaced with the I domain of alpha(M), the chimeric receptor supported cell migration to Fg; however, the alpha(M) subunit, containing the I domain but lacking the beta(2) subunit, supported migration poorly, thus, the alpha(M)I domain is necessary but not sufficient to support chemotaxis, and efficient migration requires the beta(2) subunit and alpha(M)I domain; and (c) in addition to supporting cell migration, P2 enhanced alpha(M)beta(2)-mediated chemotaxis to Fg and the P1 peptide. This activation was associated with exposure of the activation-dependent epitope recognized by monoclonal antibody 7E3 and was observed also with human neutrophils. Taken together, these data define specific molecular requirements for alpha(M)beta(2) to mediate cell migration to Fg derivatives and assign a novel proinflammatory activity to the P2 peptide.

Interaction of the fungal pathogen Candida albicans with integrin CD11b/CD18: recognition by the I domain is modulated by the lectin-like domain and the CD18 subunit.

Interactions of microorganisms with integrins are central to the host defense mechanisms. The leukocyte integrin CD11b/CD18 is the principal adhesion receptor on leukocytes for Candida albicans, a major opportunistic pathogen. In this study we have investigated the roles of three regions within the receptor, the inserted (I) and lectin-like domains within the CD11b subunit, and the CD18 subunit, in CD11b/CD18-C. albicans interactions. We report four major findings. 1) A mutation in CD18 exerts a dominant negative effect on the function of the CD11b/CD18 complex. This interpretation is based on the observation that in the absence of CD18, the CD11b subunit alone binds C. albicans well, but a single point mutation at Ser138 of CD18 abolishes CD11b/CD18 binding of the fungus. 2) The lectin-like domain is not sufficient for CD11b/CD18-C. albicans interactions. Rather, the lectin-like domain appears to influence CD11b/CD18 binding activity by modulating the function of the I domain. 3) The I domain is the primary binding site for C. albicans in the receptor and is sufficient to support an efficient interaction. 4) We have identified specific amino acid sequences within the I domain that engage the microorganism. Compared with other ligands of CD11b/CD18, C. albicans has some unique as well as common contact sites within the I domain of the receptor. Such unique contact sites may underlie the ability of C. albicans to modulate CD11b/CD18 function and raise the possibility for selective interference of the microorganism-host leukocyte interactions.

Lymphocytes utilize CD11b/CD18 for adhesion to Candida albicans.

Large granular lymphocytes require adherence to hyphae of Candida albicans to inhibit growth of this fungus. This study was undertaken to identify the lymphocyte surface structures that mediate this adhesion. Monoclonal antibodies specific for epitopes of the alpha subunit (CD11b) and the beta 2 subunit (CD18) of Mac-1 eliminated lymphocyte adhesion to C. albicans hyphae. Significant inhibition of lymphocyte adhesion to C. albicans was also achieved with known protein ligands of Mac-1. These proteins included the extracellular matrix proteins vitronectin, laminin, and fibrinogen as well as two engineered peptides containing RGD (arginine-glycine-aspartic acid) sequences. Carbohydrates including N-acetyl-D-glucosamine which have been demonstrated to inhibit Mac-l-mediated adhesion to whole yeast and yeast zymosan also blocked lymphocyte adhesion to hyphae. These results identify Mac-1 (CD11b/CD18) as the surface structure that mediates lymphocyte adhesion to C. albicans. A model is proposed for lymphocyte Mac-1 activation by microbial ligands.

A quantitative radiometric assay to measure mammalian cell binding to hyphae of Candida albicans.

A rapid and reproducible assay has been developed to measure the capacity of lymphocytes to bind to Candida albicans. Lymphocytes that bound to C. albicans were either the large granular lymphocyte cell line, YT, or interleukin-2 activated lymphocytes. Lymphocyte binding was assessed as the associated radioactivity of 51Cr-labeled lymphocytes with preformed hyphae. The assay was sensitive to detection of 0.6 lymphocytes/one hyphal form at one half maximal lymphocyte binding capacity. The assay correlated well with direct microscopic assessment of lymphocyte binding to C. albicans and provided quantitative radiometric data. Although the assay was developed for the assessment of lymphocyte adhesion to C. albicans, it can be used to measure binding of other mammalian cells (e.g., polymorphonuclear leukocytes) to this fungus. In addition, the assay may be used to identify molecules involved in the adhesion of lymphocytes and other mammalian cells to C. albicans.