Dynamics of macrophage polarization reveal new mechanism to inhibit IL-1β release through pyrophosphates

In acute inflammation, extracellular ATP activates P2X7 ion channel receptors (P2X7R) on M1 polarized macrophages to release pro-inflammatory IL-1β through activation of the caspase-1/nucleotide-binding domain and leucine-rich repeat receptor containing pyrin domain 3 (NLRP3) inflammasome. In contrast, M2 polarized macrophages are critical to the resolution of inflammation but neither actions of P2X7R on these macrophages nor mechanisms by which macrophages switch from pro-inflammatory to anti-inflammatory phenotypes are known. Here, we investigated extracellular ATP signalling over a dynamic macrophage polarity gradient from M1 through M2 phenotypes. In macrophages polarized towards, but not at, M2 phenotype, in which intracellular IL-1β remains high and the inflammasome is intact, P2X7R activation selectively uncouples to the NLRP3-inflammasome activation but not to upstream ion channel activation. In these intermediate M1/M2 polarized macrophages, extracellular ATP now acts through its pyrophosphate chains, independently of other purine receptors, to inhibit IL-1β release by other stimuli through two independent mechanisms: inhibition of ROS production and trapping of the inflammasome complex through intracellular clustering of actin filaments

AIM2 and NLRC4 inflammasomes contribute with ASC to acute brain injury independently of NLRP3

Inflammation that contributes to acute cerebrovascular disease is driven by the proinflammatory cytokine interleukin-1 and is known to exacerbate resulting injury. The activity of interleukin-1 is regulated by multimolecular protein complexes called inflammasomes. There are multiple potential inflammasomes activated in diverse diseases, yet the nature of the inflammasomes involved in brain injury is currently unknown. Here, using a rodent model of stroke, we show that the NLRC4 (NLR family, CARD domain containing 4) and AIM2 (absent in melanoma 2) inflammasomes contribute to brain injury. We also show that acute ischemic brain injury is regulated by mechanisms that require ASC (apoptosis-associated speck-like protein containing a CARD), a common adaptor protein for several inflammasomes, and that the NLRP3 (NLR family, pyrin domain containing 3) inflammasome is not involved in this process. These discoveries identify the NLRC4 and AIM2 inflammasomes as potential therapeutic targets for stroke and provide new insights into how the inflammatory response is regulated after an acute injury to the brain

Estúdio de la reacción entre el Ce (III) y el Acido Trietilentetraaminohexaacético. Determinación espectrofotométrica de cerio

A spectrophotometric study about the reaction between Ce(III) and triethylentetraaminehexaacetic acid (TTHA) is presented. At pH 5 Ce(III) forms with TTHA a 1:1 molar ratio complex which shows an absorption maximum at 280 nm (ε = 824 mol-1 . cm-1 . 1) and is suitable for the spectrophotometric determination of cerium (15-150 p. p. m.). The standard deviation for ten determinations at 1,97 mg leve1 was 1,97 . 10-3.Se presenta un estudio espectrofotométrico de la reacción entre el Ce(III) y el ácido trietilentetraaminohexaacético (ATTH). A pH 5 el Ce(III) forma con el ATTH un complejo de relación molar 1:1 que presenta un máximo de absorción a 280 nm ε = 824 mol-1 . cm-1.1) y es adecuado para la determinación espectrofotométrica de cerio (15-150 p. p. m.). La desviación estándar para diez determinaciones (1,97 mg de Ce) fue 2,79 . 10-3

P2X receptors: epithelial ion channels and regulators of salt and water transport.

When the results from electrophysiological studies of renal epithelial cells are combined with data from in vivo tubule microperfusion experiments and immunohistochemical surveys of the nephron, the accumulated evidence suggests that ATP-gated ion channels, P2X receptors, play a specialized role in the regulation of ion and water movement across the renal tubule and are integral to electrolyte and fluid homeostasis. In this short review, we discuss the concept of P2X receptors as regulators of salt and water salvage pathways, as well as acknowledging their accepted role as ATP-gated ion channels

Differential IL-1β secretion by monocyte subsets is regulated by Hsp27 through modulating mRNA stability.

Monocytes play a central role in regulating inflammation in response to infection or injury, and during auto-inflammatory diseases. Human blood contains classical, intermediate and non-classical monocyte subsets that each express characteristic patterns of cell surface CD16 and CD14; each subset also has specific functional properties, but the mechanisms underlying many of their distinctive features are undefined. Of particular interest is how monocyte subsets regulate secretion of the apical pro-inflammatory cytokine IL-1β, which is central to the initiation of immune responses but is also implicated in the pathology of various auto-immune/auto-inflammatory conditions. Here we show that primary human non-classical monocytes, exposed to LPS or LPS + BzATP (3'-O-(4-benzoyl)benzyl-ATP, a P2X7R agonist), produce approx. 80% less IL-1β than intermediate or classical monocytes. Despite their low CD14 expression, LPS-sensing, caspase-1 activation and P2X7R activity were comparable in non-classical monocytes to other subsets: their diminished ability to produce IL-1β instead arose from 50% increased IL-1β mRNA decay rates, mediated by Hsp27. These findings identify the Hsp27 pathway as a novel therapeutic target for the management of conditions featuring dysregulated IL-1β production, and represent an advancement in understanding of both physiological inflammatory responses and the pathogenesis of inflammatory diseases involving monocyte-derived IL-1β

The Second Transmembrane Domain of P2X7 Contributes to Dilated Pore Formation

Activation of the purinergic receptor P2X7 leads to the cellular permeability of low molecular weight cations. To determine which domains of P2X7 are necessary for this permeability, we exchanged either the C-terminus or portions of the second transmembrane domain (TM2) with those in P2X1 or P2X4. Replacement of the C-terminus of P2X7 with either P2X1 or P2X4 prevented surface expression of the chimeric receptor. Similarly, chimeric P2X7 containing TM2 from P2X1 or P2X4 had reduced surface expression and no permeability to cationic dyes. Exchanging the N-terminal 10 residues or C-terminal 14 residues of the P2X7 TM2 with the corresponding region of P2X1 TM2 partially restored surface expression and limited pore permeability. To further probe TM2 structure, we replaced single residues in P2X7 TM2 with those in P2X1 or P2X4. We identified multiple substitutions that drastically changed pore permeability without altering surface expression. Three substitutions (Q332P, Y336T, and Y343L) individually reduced pore formation as indicated by decreased dye uptake and also reduced membrane blebbing in response to ATP exposure. Three others substitutions, V335T, S342G, and S342A each enhanced dye uptake, membrane blebbing and cell death. Our results demonstrate a critical role for the TM2 domain of P2X7 in receptor function, and provide a structural basis for differences between purinergic receptors. © 2013 Sun et al

Genomic Organization, Splice Variants and Expression of CGMl, a CD66-related Member of the Carcinoembryonic Antigen Gene Family

The tumor marker carcinoembryonic antigen (CEA) belongs to a family of proteins which are composed of one immunogiobulin variable domain and a varying number of immunoglobulin constant-like domains. Most of the membrane-bound members, which are anchored either by a glycosylphosphatidylinositol moiety or a transmembrane domain, have been shown to convey cell adhesion in vitro. Here we describe two splice variants of CGMI. a transmembrane member of the CEA family without immunoglobulin constant.like domains. CGM1a and CGM1c contain cytopiasmic domains of 71 and 31 amino acids, respectively, The cytoplasmic region of CGM1a is encoded by four exons (Cyt1-Cyt4). Differential splicing of the Cyt1 exon (53 bp)..

Intestinal secretory and absorptive functions in Trichinella spiralis mouse model of postinfective gut dysfunction: role of bile acids

ABSTRACT Objective: Observations showing that bile acid malabsorption is frequent in irritable bowel syndrome (IBS) suggest that alterations in bile acid-induced secretion and absorption could contribute to IBS-associated diarrhoea. The secretory response to bile acids, fluid transport and bile absorption was examined in intestinal tissues from a Trichinella spiralis mouse model of postinfectious gut dysfunction in vitro. Changes in the protein expression of apical sodium-dependent bile acid transporter (ASBT) were also measured. Design: T. spiralis-infected mice were killed at 18 and 25 days postinfection. Jejunal, ileal, proximal and distal colon segments were exposed to taurodeoxycholic acid (TDCA) or cholic acid. Short circuit current (SCC) increases were determined. Tritiated taurocholic acid (3H-TCA) absorption was determined in everted jejunal and ileal sacs. ASBT protein expression was determined by Western blot analysis and immunohistochemistry. Results: Basal SCC increased in ileum and distal colon at 18 and 25 days postinfection, respectively. Ileal SCC responses to TDCA and cholic acid were enhanced at 18 days postinfection. Distal colon SCC response to TDCA was raised at 18 days postinfection but was significantly reduced by 25 days. Ileal 3H-TCA uptake was significantly reduced at 18 and 25 days postinfection. Surprisingly, increased ASBT expression was observed in infected animals. Conclusions: In a T. spiralis model of postinfectious gut dysfunction, decreased bile absorption and enhanced secretion in response to bile acids was observed. Decreased absorption was not, however, caused by decreased ASBT as increased expression was observed. If similar events occur postinfection, the combined effects of these disturbances may contribute to some symptoms observed in postinfectious IBS patients. Irritable bowel syndrome (IBS) is an extremely common disorder that affects up to 20% of the general population and is responsible for almost half of the referrals to gastroenterologists. 1 2 Surprisingly, the cause of IBS is poorly understood and several pathophysiological mechanisms have been implicated. [3] 11 Although bile acid malabsorption (BAM) has generally been regarded an infrequent cause of chronic diarrhoea, recent improvements in the techniques employed for assessing BAM have demonstrated that it is a much more common cause of diarrhoea than originally considered. 12 13 This has been highlighted by the recent and unexpected evidence that BAM was observed in 33% of patients with diarrhoea-predominant IBS. 13 Bile acids are synthesised in the liver and secreted into the small intestine where they facilitate fat and fat-soluble vitamin absorption. Although some bile uptake occurs in the jejunum, the main route for circulation of bile back into the liver is by active reabsorption in the terminal ileum by the apical sodium-dependent bile acid transporter (ASBT). Dysfunction of ASBT is accompanied by an interruption in the enterohepatic bile circulation, allowing bile acids to enter the colon in increased concentrations. 14 15 This subsequently induces diarrhoea as bile acids stimulate chloride ion (Cl 2 ) secretion and powerful propagating contractions in the colon. 16 Although IBS has generally been considered a motility disorder, it seems likely that the condition may involve changes in fluid and electrolyte transport across the intestinal epithelium, because diarrhoea and mucus hypersecretion are well-recognised features. In addition, intestinal secretory mechanisms may be more sensitive to secretogogues, such as bile acids, during IBS. Oddsson and colleagues 17 have shown that the small intestine in IBS patients has a greater secretory response to low bile acid concentrations. Recent work in our laboratory and by others has established the characteristics of bile acid-induced secretion and ileal bile acid absorption in normal and mast cell-deficient mice. 18-21 Similar studies have not been performed in a murine model of postinfectious gut dysfunction. The T. spiralisinfected mouse is a widely acknowledged model of postinfectious gut dysfunction in which visceral hypersensitivity and persistently altered motility, which mimic the hyperreactive state in IBS, are observed. 10 22 T. spiralis infection has two phases. Postinfectious gut dysfunction occurs after the enteric phase, when the worm is expelled from the intestine. There is also a skeletal muscle phase during which the worm is present in muscle (for the duration of the mouse's life) despite gut expulsion. Although infection with the nematode initially generates an intestinal inflammatory response that resolves after worm expulsion from the intestine, functional changes such as increased motility, visceral hypersensitivitity and increased muscle thickness persist. The current study determined the secretory effects of cholic acid and taurodeoxycholic acid (TDCA) in the small intestine and colon of T. spiralis-infected mice at two postinfective timepoints. In addition, studies were performed to investigate whether passive bile uptake in the jejunum and active bile absorption in the terminal ileum was also impaired in these mice. Furthermore, changes in the expression of ASBT after infection were determined in an attempt to correlate these with any observed differences in bile salt absorption. MATERIALS AND METHODS Animals Experiments were performed on intestinal tissues from T. spiralis-infected and non-infected mice killed by cervical dislocation in accordance with UK Home Office regulations and with local Ethical Committee approval. Male Swiss mice (age 12-13 weeks) were obtained from Sheffield Field Laboratories and were allowed free access to food and water. Infection with T. spiralis Stock mice infected with T. spiralis were killed to obtain larvae for infecting mice to be used in experimental procedures. Larvae were recovered from stock mice by pepsin (0.5%) and hydrochloric acid (0.5%) digestion of the skeletal muscle as described by Castro and Fairbain. Measurement of transintestinal electrical activity Ussing chambers were used to measure changes in ion transport through the electrical correlate, short circuit current (SCC). Segments of jejunum (immediately distal to the ligament of Treitz), terminal ileum (6 cm before the caecum), proximal colon and distal colon were stripped of the outer muscle layers, which removed the myenteric plexus as well as the muscle coat but left intact the submucosal and mucosal plexus. Tissue was allowed to stabilise for 15 minutes after mounting, and readings of electrical activity were subsequently taken at one minute intervals. After five minutes of basal readings, either cholic acid (Sigma, St Louis, Missouri, USA) or TDCA (Sigma) was added to the serosal side and readings were taken for a further 15 minutes. We have previously looked at the effects of both the mucosal and serosal application of several bile acids to the small intestine and found a concentration of 1 mmol to be effective only from the serosal side. 19 Therefore bile acid secretion is initiated by action at the serosal side of the enterocyte. The actual effective concentration is likely to be considerably less because of the diffusion barrier, represented by subepithelial tissues, which needs to be overcome. Furthermore, in the ileum, sodium-dependent bile acid absorption also increases SCC, so to avoid this component of the overall SCC change that occurs when bile acids are applied mucosally, serosal application (when absorption is not activated) was chosen. An aliquot of 100 ml cholic acid or TDCA, dissolved in ethanol and saline, respectively, was added to the 5 ml bathing solution to yield a final concentration of 1 mmol for both substances. Preliminary studies identified that neither ethanol nor saline, added serosally, had any significant effect on basal electrical activity. The SCC generated by the sheets after bile acid administration was calculated as described above using Ohm's law. Measurement of intestinal fluid transport The transport of fluid by the mucosa was measured in everted sacs taken from proximal jejunum and terminal ileum. A 5-7 cm intestinal segment, everted on a glass rod, was filled with 0.2 ml Krebs bicarbonate saline containing 10 mmol glucose (serosal fluid) and was incubated for 30 minutes in 15 ml Krebs bicarbonate saline containing 10 mmol mannitol (mucosal fluid) at 37uC in a shaking water bath. Results are expressed as mucosal fluid transport (MFT), which is the sum of the increase in the volume (weight of tissue) of serosal fluid in the sac after incubation (serosal fluid transport) and that taken up by the gut itself (gut fluid uptake) and values were related to the initial wet weight of the empty sac (ml/g initial wet weight/ 30 minutes). Measurement of 3H-TCA absorption The absorption of taurocholic acid (TCA) was also assessed in the same everted sacs by adding TCA (1 mmol; Sigma) together with 3H-TCA (2.5 mCi/100 ml; PerkinElmer Life Sciences, Boston, Massachusetts, USA) to the mucosal fluid. At the end of the incubation period the serosal fluid was collected. The sac was deproteinised using 10% sodium tungstate (1.25 ml) and 0.33 MH 2 SO 4 (1.25 ml), homogenised and then filtered. Scintillation fluid (3 ml; Emulsifer-safe; Packard Biosciences, USA) was added to 100 ml samples of initial mucosal fluid, final mucosal fluid, final serosal fluid and gut homogenate, and radioactivity was determined using a liquid scintillation analyser (Packard TRI-CARB, 1900XR; Packard Biosciences, Pangbourne, Berkshire, UK). TCA absorption was expressed in two ways: first, as the amount taken up by the sac (mmol/g initial wet weight/30 minutes) and second as the T/M ratio, i.e. the ratio of the TCA concentration in the tissue water compared with its concentration in the mucosal fluid at the end of the incubation period, whereby a T/M ratio greater than 1 indicated active transport. Intestinal inflammation Preparation of epithelial cell homogenates for ASBT expression studies Epithelial cell homogenates were prepared from the three contiguous 3 cm segments of the most distal part of the small intestine. All further steps were performed with the preparations kept on ice. Segments were opened in the longitudinal axis and washed in isotonic saline (0.9% NaCl) to discard adhering luminal content. Mucosa were scraped with a clean glass rod. The mucosal scrapings of three animals were suspended in 5 ml buffer A (10 mmol Tris/HCl/0.13 mol NaCl/5 mmol EDTA, pH 7.4) and stirred gently for 30 minutes at 4uC. Cells were collected by centrifugation (3 min, 2000 rpm, 4uC) and suspended in 500 ml buffer B (10 mmol Tris/HCl/0.3 mol mannitol, pH 7.2) and 20 ml of a protease inhibitor cocktail (Roche, Hertfordshire, UK) to a concentration of approximately 1-2 mg/ ml. The samples containing proteins were stored at 220uC until use. The protein concentration was determined by the Bradford method (Biorad Laboratories, Munchen, Germany). Samples were solubilised in 56 sodium dodecylsulfate (SDS) sample buffer containing 0.125 mmol Tris?Cl, pH 6.8, 10% SDS, 50% glycerol, 10% mercaptoethanol and 0.005% bromophenol blue, and then boiled at 100uC for 5 minutes