DNA attenuates enterocyte Toll-like receptor 4-mediated intestinal mucosal injury after remote trauma

Intestinal mucosal injury occurs after remote trauma although the mechanisms that sense remote injury and lead to intestinal epithelial disruption remain incompletely understood. We now hypothesize that Toll-like receptor 4 (TLR4) signaling on enterocytes after remote injury, potentially through the endogenous TLR4 ligand high-mobility group box-1 (HMGB1), could lead to intestinal dysfunction and bacterial translocation and that activation of TLR9 with DNA could reverse these effects. In support of this hypothesis, exposure of TLR4-expressing mice to bilateral femur fracture and systemic hypotension resulted in increased TLR4 expression and signaling and disruption of the ileal mucosa, leading to bacterial translocation, which was not observed in TLR4-mutant mice. TLR4 signaling in enterocytes, not immune cells, was required for this effect, as adenoviral-mediated inhibition of TLR4 in enterocytes prevented these findings. In seeking to identify the endogenous TLR4 ligands involved, the expression of HMGB1 was increased in the intestinal mucosa after injury in wild-type, but not TLR4-mutant, mice, and administration of anti-HMGB1 antibodies reduced both intestinal mucosal TLR4 signaling and bacterial translocation after remote trauma. Strikingly, mucosal injury was significantly increased in TLR9-mutant mice, whereas administration of exogenous DNA reduced the extent of TLR4-mediated enterocyte apoptosis, restored mucosal healing, and maintained the histological integrity of the intestinal barrier after remote injury. Taken together, these findings identify a novel link between remote injury and enterocyte TLR4 signaling leading to barrier injury, potentially through HMGB1 as a ligand, and demonstrate the reversal of these adverse effects through activation of TLR9.     

Hemorrhagic shock induces NAD(P)H oxidase activation in neutrophils: role of HMGB1-TLR4 signaling

Hemorrhagic shock/resuscitation (HS/R)-induced generation of reactive oxygen species (ROS) plays an important role in posthemorrhage inflammation and tissue injury. We have recently reported that HS/R-activated neutrophils (PMN), through release of ROS, serve an important signaling function in mediating alveolar macrophage priming and lung inflammation. PMN NAD(P)H oxidase has been thought to be an important source of ROS following HS/R. TLR4 sits at the interface of microbial and sterile inflammation by mediating responses to both bacterial endotoxin and multiple endogenous ligands, including high-mobility group box 1 (HMGB1). Recent studies have implicated HMGB1 as an early mediator of inflammation after HS/R and organ ischemia/reperfusion. In the present study, we tested the hypothesis that HS/R activates NAD(P)H oxidase in PMN through HMGB1/TLR4 signaling. We demonstrated that HS/R induced PMN NAD(P)H oxidase activation, in the form of phosphorylation of p47phox subunit of NAD(P)H oxidase, in wild-type mice; this induction was significantly diminished in TLR4-mutant C3H/HeJ mice. HMGB1 levels in lungs, liver, and serum were increased as early as 2 h after HS/R. Neutralizing Ab to HMGB1 prevented HS/R-induced phosphorylation of p47phox in PMN. In addition, in vitro stimulation of PMN with recombinant HMGB1 caused TLR4-dependent activation of NAD(P)H oxidase as well as increased ROS production through both MyD88-IRAK4-p38 MAPK and MyD88-IRAK4-Akt signaling pathways. Thus, PMN NAD(P)H oxidase activation, induced by HS/R and as mediated by HMGB1/TLR4 signaling, is an important mechanism responsible for PMN-mediated inflammation and organ injury after hemorrhage.     

Thioredoxin catalyzes the denitrosation of low-molecular mass and protein S-nitrosothiols

While most proteins have critical thiols whose oxidation affects their activity, it has been suggested that S-nitrosation and denitrosation of cellular thiols are fundamental processes similar to protein phosphorylation and dephosphorylation, respectively. However, understanding the biosynthesis and catabolism of S-nitrosothiols has proven to be difficult, in part because of the low stability of this class of metabolites. Herein, we report that thioredoxin catalyzes the denitrosation of a series of S-nitrosoamino acids and S-nitrosoproteins derived from HepG2 cells. Notably, all S-nitrosoproteins with a molecular mass of 23-30 kDa were catabolized by thioredoxin. Experimental evidence is presented which shows that both glutathione and reduced human thioredoxin denitrosate S-nitrosothioredoxin, which has been suggested to act as an anti-apoptotic factor via trans-S-nitrosation of caspase 3. In HepG2 cells, we observed that S-nitrosocysteine ethyl ester impedes the activity of caspase 3. However, a subsequent incubation of the cells in nitrosothiol-free medium resulted in reconstitution of the enzymatic activity, most likely due to endogenous denitrosation of S-nitrosocaspase 3. The latter process was markedly inhibited in thioredoxin reductase-deficient HepG2 cells, suggesting that the thioredoxin/thioredoxin reductase system tends to maintain intracellular caspase 3 in a reduced, SH state. The data obtained are discussed within the general reaction mechanisms encompassing the cellular homeostasis of S-nitrosothiols.     

The Effect of Substrate Stiffness,Thickness, and Cross-Linking Density on Osteogenic Cell Behavior

Osteogenic cells respond to mechanical changes in their environment by altering their spread area, morphology, and gene expression profile. In particular, the bulk modulus of the substrate, as well as its microstructure and thickness, can substantially alter the local stiffness experienced by the cell. Although bone tissue regeneration strategies involve culture of bone cells on various biomaterial scaffolds, which are often cross-linked to enhance their physical integrity, it is difficult to ascertain and compare the local stiffness experienced by cells cultured on different biomaterials. In this study, we seek to characterize the local stiffness at the cellular level for MC3T3-E1 cells plated on biomaterial substrates of varying modulus, thickness, and cross-linking concentration. Cells were cultured on flat and wedge-shaped gels made from polyacrylamide or cross-linked collagen. The cross-linking density of the collagen gels was varied to investigate the effect of fiber cross-linking in conjunction with substrate thickness. Cell spread area was used as a measure of osteogenic differentiation. Finite element simulations were used to examine the effects of fiber cross-linking and substrate thickness on the resistance of the gel to cellular forces, corresponding to the equivalent shear stiffness for the gel structure in the region directly surrounding the cell. The results of this study show that MC3T3 cells cultured on a soft fibrous substrate attain the same spread cell area as those cultured on a much higher modulus, but nonfibrous substrate. Finite element simulations predict that a dramatic increase in the equivalent shear stiffness of fibrous collagen gels occurs as cross-linking density is increased, with equivalent stiffness also increasing as gel thickness is decreased. These results provide an insight into the response of osteogenic cells to individual substrate parameters and have the potential to inform future bone tissue regeneration strategies that can optimize the equivalent stiffness experienced by a cell.     

High Mobility Group Box 1 (HMGB1) Phenotypic Role Revealed with Stress

High mobility group box 1 (HMGB1) is an evolutionarily ancient protein that is present in one form or another in all eukaryotes. It fundamentally resides in the nucleus but translocates to the cytosol with stress and is subsequently released into the extracellular space. HMGB1 global knockout mice exhibit lethal hypoglycemia, whereas tissues and cells from conditional knockout or knock-in mice are born alive without apparent significant functional deficit. An aberrant response to targeted stress in the liver, pancreas, heart or myeloid cells is consistent with a protective role for HMGB1 in sustaining nuclear homeostasis and enabling other stress responses, including autophagy. Under some conditions, HMGB1 is not required for liver and heart function. Many challenges remain with respect to understanding the multiple roles of HMGB1 in health and disease.     

Cell Death and DAMPs in Acute Pancreatitis

Cell death and inflammation are key pathologic responses of acute pancreatitis (AP), the leading cause of hospital admissions for gastrointestinal disorders. It is becoming increasingly clear that damage-associated molecular pattern molecules (DAMPs) play an important role in the pathogenesis of AP by linking local tissue damage to systemic inflammation syndrome. Endogenous DAMPs released from dead, dying or injured cells initiate and extend sterile inflammation via specific pattern recognition receptors. Inhibition of the release and activity of DAMPs (for example, high mobility group box 1, DNA, histones and adenosine triphosphate) provides significant protection against experimental AP. Moreover, increased serum levels of DAMPs in patients with AP correlate with disease severity. These findings provide novel insight into the mechanism, diagnosis and management of AP. DAMPs might be an attractive therapeutic target in AP.     

River macrophyte indices: not the Holy Grail!

1. Recent studies have demonstrated that there is generally no unambiguous relationship between plant species composition and specific environmental conditions in rivers. Nevertheless, indices of environmental pressures based on macrophytes are flourishing, because of the requirements of the Water Framework Directive (WFD). 2. We first reviewed nine such indices against 13 criteria for bioindicators. Then, using data from France and England, we tested whether the IBMR (Macrophyte Biological Index for Rivers) and LEAFPACS (predictions and classification system for macrophytes) methods could reliably indicate nutrient and hydromorphological pressures. Finally, we used an improved bootstrapping method to estimate accuracy. 3. Currently, most indices lack ecological meaning for a variety of reasons, including partial sampling (backwaters are excluded); reliance on list of taxa (there are identification difficulties) rather than structure and functions; correlation rather than causation; application within a limited biogeographical area; reliance on ‘expert’ judgement; high precision but poor accuracy; poorly defined reference conditions; lack of independent tests; and an inability to discriminate reliably between the target pressures of interest from confounding background variables. 4. IBMR was a far better indicator of pH (or HCO₃‐pCO₂) than it was of soluble reactive phosphorus, SRP (or SRP‐NH₄). While there was a highly significant correlation between IBMR and SRP after removing the effect of pH, the relationship was weak (r² = 0.08, n = 215, P < 0.001). 5. LEAFPACS is a multi‐metric method summing up five individual indices, each compliant with the WFD. Its individual metrics were not better correlated with nutrient and hydromorphological pressures (with r² < 0.1, n = 62, P < 0.05) than was the IBMR. The meaning of the overall metric is questionable. 6. There are problems in determining the precision of the indices, owing to uncertainties in recording, but they are less than the uncertainties in determining accuracy (because species optima and tolerances are sometimes poorly known). 7. Reliable information is needed to improve the state of our rivers. Macrophyte indices are able to detect statistically significant pressures from a large population of sites but cannot be applied at specific sites, as required by the WFD, owing to large uncertainties and low explanatory power. Typically, more than 90% of the variability in macrophyte indices is attributed to factors other than human pressure. The WFD would be better served by a simpler, holistic approach based on our current mechanistic understanding of river processes. These findings are likely to apply also to other taxonomic groups (macroinvertebrates, diatoms, fish) used in the assessment of purported ecological quality and to palaeolimnological measures of reference status.     

Using in vivo Cine and 3D multi-contrast MRI to determine human atherosclerotic carotid artery material properties and circumferential shrinkage rate and their impact on stress/strain predictions

In vivo magnetic resonance image (MRI)-based computational models have been introduced to calculate atherosclerotic plaque stress and strain conditions for possible rupture predictions. However, patient-specific vessel material properties are lacking in those models, which affects the accuracy of their stress/strain predictions. A noninvasive approach of combining in vivo Cine MRI, multicontrast 3D MRI, and computational modeling was introduced to quantify patient-specific carotid artery material properties and the circumferential shrinkage rate between vessel in vivo and zero-pressure geometries. In vivo Cine and 3D multicontrast MRI carotid plaque data were acquired from 12 patients after informed consent. For each patient, one nearly-circular slice and an iterative procedure were used to quantify parameter values in the modified Mooney-Rivlin model for the vessel and the vessel circumferential shrinkage rate. A sample artery slice with and without a lipid core and three material parameter sets representing stiff, median, and soft materials from our patient data were used to demonstrate the effect of material stiffness and circumferential shrinkage process on stress/strain predictions. Parameter values of the Mooney-Rivlin models for the 12 patients were quantified. The effective Young's modulus (YM, unit: kPa) values varied from 137 (soft), 431 (median), to 1435 (stiff), and corresponding circumferential shrinkages were 32%, 12.6%, and 6%, respectively. Using the sample slice without the lipid core, the maximum plaque stress values (unit: kPa) from the soft and median materials were 153.3 and 96.2, which are 67.7% and 5% higher than that (91.4) from the stiff material, while the maximum plaque strain values from the soft and median materials were 0.71 and 0.293, which are about 700% and 230% higher than that (0.089) from the stiff material, respectively. Without circumferential shrinkages, the maximum plaque stress values (unit: kPa) from the soft, median, and stiff models were inflated to 330.7, 159.2, and 103.6, which were 116%, 65%, and 13% higher than those from models with proper shrinkage. The effective Young's modulus from the 12 human carotid arteries studied varied from 137 kPa to 1435 kPa. The vessel circumferential shrinkage to the zero-pressure condition varied from 6% to 32%. The inclusion of proper shrinkage in models based on in vivo geometry is necessary to avoid over-estimating the stresses and strains by up 100%. Material stiffness had a greater impact on strain (up to 700%) than on stress (up to 70%) predictions. Accurate patient-specific material properties and circumferential shrinkage could considerably improve the accuracy of in vivo MRI-based computational stress/strain predictions.     

A Janus Tale of Two Active High Mobility Group Box 1 (HMGB1) Redox States

High mobility group box 1 (HMGB1), the prototypic damage–associated molecular pattern molecule, is released at sites of inflammation and/or tissue damage. There, it promotes cytokine production and chemokine production/cell migration. New work shows that the redox status of HMGB1 distinguishes its cytokine-inducing and chemokine activity. Reduced all-thiol-HMGB1 has sole chemokine activity, whereas disulfide-HMGB1 has only cytokine activity, and oxidized, denatured HMGB1 has neither. Autophagy (programmed cell survival) and apoptosis (programmed cell death) have been implicated in controlling both innate and adaptive immune functions. Reduced HMGB1 protein promotes autophagy, whereas oxidized HMGB1 promotes apoptosis. Thus, the differential activity of HMGB1 in immunity, inflammation and cell death depends on the cellular redox status within tissues.High mobility group box 1 (HMGB1), a nonhistone nuclear factor, acts extracellularly as a damage-associated molecular pattern (DAMP) molecule to modulate inflammation, promoting autophagy and innate immune responses (1–5). HMGB1 has compartment-specific functions: nuclear, intracellular (but extranuclear) and extracellular. Its extracellular functions can now be divided further into cytokine-like or cytokine-inducing, chemokinelike and proangiogenic. Signaling pathways that induce variations on the posttranslational modification, such as phosphorylation and acetylation, have been implicated in the regulation of HMGB1 release. Importantly, HMGB1 contains three cysteines, each of which is susceptible to redox modification (6,7). The redox state of these cysteines is important for the proinflammatory cytokine-stimulating and proautophagic activity of HMGB1 (8–10). Autophagy (literally “self-eating”), a lysosome-mediated catabolic process, contributes to maintenance of intracellular homeostasis and promotes cell survival in response to environmental stress (11–13).Treatment with reduced but not oxidized HMGB1 protein increases autophagy in cancer cells (9). In contrast, oxidized HMGB1 protein activates the caspase-dependent apoptotic cell death pathway (9). Venereau et al.(14) described a new role for redox control of both the cytokine-inducing and chemokine activity of HMGB1 in the setting of sterile inflammation, regulating leukocyte recruitment and their ability to secrete inflammatory cytokines (Figure 1).Open in a separate windowFigure 1Redox control of HMGB1 activity. To act as a DAMP/danger signal and inflammatory mediator, HMGB1 is transported extracellularly by two principal means: active secretion from living inflammatory cells (for example, macrophages) or passive release from necrotic cells. The activities of extracellular HMGB1 are redox dependent. All-thiol-HMGB1 promotes chemokine production and leukocyte recruitment. Disulfide-HMGB1, originating from infiltrating leukocytes, promotes release of proinflammatory cytokines and thus participates in the inflammatory response. Reactive oxygen species produced by leukocytes induces the terminal oxidation of HMGB1, which is inactivated during resolution of inflammation.Structurally, HMGB1 is composed of three domains: two positively charged proximal DNA-binding domains (A box and B box) and a negatively charged carboxyl terminus. Three cysteines are encoded within the molecule: two vicinal cysteines in box A (C23 and C45) and a single one in box B (C106). C23 and C45 can form an intermolecular disulfide bond, whereas C106 is unpaired. Therefore, three different redox forms HMGB1 (all-thiol-HMGB1, disulfide-HMGB1 and oxidized HMGB1) were derived from bacterial expression systems (14). In addition, by using tryptic digests and liquid chromatography tandem mass spectrometric analysis, Venereau et al. observed that recombinant HMGB1 can be reversibly oxidized and reduced in the presence of electron donors (for example, dithiothreitol) or acceptors (oxygen) (14).Next, Venereau et al. assessed whether individual redox forms of HMGB1 have a differential role in cytokine-stimulating and chemoattractant activities (14). They found that disulfide-HMGB1 induced activation of the nuclear factor (NF)-κB pathway and production of proinflammatory cytokines (for example, tumor necrosis factors-α, interleukin [IL]-6 and IL-8) in fibroblasts and macrophages. Interestingly, all-thiol-HMGB1 failed to induce a proinflammatory response. In contrast, all-thiol-HMGB1, but not disulfide-HMGB1, had chemoattractant activity in fibroblasts. These findings prompted them to determine whether HMGB1 inhibitors, such as box A and monoclonal antibody PDH1.1, block the chemoattractant and/or cytokine-inducing activities of HMGB1. Unexpectedly, these inhibitors prevented cell migration but not cytokine production, although they are widely used as HMGB1-targeting agents in experimental inflammatory diseases.Reactive oxygen species oxidize the HMGB1 released from dying cells, thereby neutralizing its stimulatory activity and promoting tolerance in immune cells (15,16). In addition, oxidation of C106 or lack of a disulfide bridge between C23 and C45 then causes HMGB1 to lose its proinflammatory effects in macrophages (8). Venereau et al. found that terminal oxidation by hydrogen peroxide results in the loss of both the cytokine-stimulating and chemoattractant activities of HMBG1. Moreover, the authors found that the three HMGB1 cysteine residues were required for the cytokine-stimulating activity but not for the chemoattractant activity of HMGB1. Cysteine mutant HMGB1 promotes fibroblast migration, but not cytokine expression in macrophages (14). Collectively, these findings establish a crucial role for redox in the regulation of HMGB1 activity in inflammation and migration.What is the redox state of HMGB1 in the pathogenesis of individual diseases? The redox state of HMGB1 from the human acute monocytic leukemia cell line THP-1 was measured in the presence or absence of lipopolysaccharide (LPS) and necrotic medium in vitro. Intracellular HMGB1 was all-thiol-HMGB1, whereas secreted HMGB1 contained both all-thiol- and disulfide-HMGB1 (14). Furthermore, disulfide-HMGB1 was present later and time-dependently increased in cardiotoxin-injured muscles in vivo, confirming that the redox state of HMGB1 is altered during tissue damage and inflammation. HMGB1 protein with all three cysteines mutated to serine are resistant to oxidation and induce leukocyte recruitment without inducing cytokine production (14). The activities of HMGB1 are thus redox-dependent and can be modified within the injured tissues after HMGB1 release. Therefore, release of dynamic redox-regulated HMGB1 contributes to the orderly orchestrated recruitment of leukocytes, activation of cytokine release and subsequent resolution of inflammation.Several issues remain unresolved regarding the redox control of HMGB1 activity. First, HMGB1 is specifically recognized by several cell surface receptors (2), including Toll-like receptor (TLR)-4 and the receptor for advanced glycation end products (RAGE), but most recently was joined by T-cell immunoglobulin and mucin domain 3 (TIM-3) (17). Initial studies suggest that reduced C106 is necessary for the binding of HMGB1 to one of its receptors, TLR4, to stimulate cytokine release (8). HMGB1-induced recruitment of inflammatory cells depends on forming a complex with CXCL12 and signaling via CXCR4 (18). Moreover, RAGE is required for reduced HMGB1-mediated autophagy, but not oxidized HMGB1-induced apoptosis (9). All-thiol-HMGB1, but not disulfide-HMGB1, binds CXCL12 (14). The influence of HMGB1 receptors (for example, RAGE, TLR4, TLR2, CD24, TIM-3 and triggering receptor expressed on myeloid cells 1 [TREM1]) on biological activities of individual redox forms of HMGB1 remains to be carefully investigated. Second, HMGB1 forms highly inflammatory complexes with DNA, lipoteichoic acid, LPS, IL-1β, chemokine (C-X-C motif) ligand 12 (CXCL12)/ stromal cell–derived factor-1 (SDF-1) and nucleosomes (19). There is great interest in determining whether the individual redox forms of HMGB1 have varying affinity profiles active in inflammation and immunity. Third, HMGB1 has multiple intracellular and extracellular functions in health and disease, including cancer (1,2,6,20). Additional studies will be needed to determine whether redox is required for other functions of HMGB1, such as regeneration and cellular differentiation as well as the complex interactions between autophagy and immunity (5). One additional unanswered question is where and how the formation of the disulfide takes place and whether there is an enzyme specific for regulating this. This is important, knowing that the nuclear form is mostly all thiol. Finally, the development and performance of a simple, sensitive method for the detection of individual HMGB1 redox state isoforms in clinical specimens remains to be accomplished.