Biology,role and therapeutic potential of circulating histones in acute inflammatory disorders

Histones are positively charged nuclear proteins that facilitate packaging of DNA into nucleosomes common to all eukaryotic cells. Upon cell injury or cell signalling processes, histones are released passively through cell necrosis or actively from immune cells as part of extracellular traps. Extracellular histones function as microbicidal proteins and are pro‐thrombotic, limiting spread of infection or isolating areas of injury to allow for immune cell infiltration, clearance of infection and initiation of tissue regeneration and repair. Histone toxicity, however, is not specific to microbes and contributes to tissue and end‐organ injury, which in cases of systemic inflammation may lead to organ failure and death. This review details the processes of histones release in acute inflammation, the mechanisms of histone‐related tissue toxicity and current and future strategies for therapy targeting histones in acute inflammatory diseases.     

重组人组蛋白H3和H4的表达纯化及其细胞毒性分析

细胞外组蛋白在脓毒症、类风湿性关节炎、急性肺损伤等多种疾病的发生发展中起关键作用,但由于缺乏合适的标准品,至今无法对患者体内的胞外组蛋白进行精确定量,导致在多种感染性疾病中无法根据血清组蛋白含量对疾病进行精确分级,也无法据此合理用药。同时,对患者体内胞外组蛋白精确定量也有助于确定细胞毒性机制研究的使用剂量。本研究用大肠杆菌表达单体变性组蛋白H3和H4,亲和纯化后用梯度稀释和透析方法,可以得到复性的组蛋白单体H3、H4以及H3/H4复合物。通过对蛋白质在纯化过程中稳定性的比较,发现H3/H4复合物较单体更为稳定。 以该复合物（50 μg/mL）处理HUVEC细胞,细胞存活率约为20%,与小牛胸腺组蛋白（200 μg/mL）的毒性类似。 该复合物引起的细胞毒性可被人血清白蛋白以浓度依赖的形式（0.625~10 mg/mL）缓解,提示其构象基本正确。 因此,重组组蛋白H3/H4复合物可以作为精确定量组蛋白的标准品,对基于组蛋白含量的疾病分级和组蛋白毒性机制的研究均有应用价值。     

TLR2 synergizes with both TLR4 and TLR9 for induction of the MyD88-dependent splenic cytokine and chemokine response to Streptococcus pneumoniae

We previously demonstrated that induction of splenic cytokine and chemokine secretion in response to Streptococcus pneumoniae (Pn) is MyD88-, but not critically TLR2-dependent, suggesting a role for additional TLRs. In this study, we investigated the role of TLR2, TLR4, and/or TLR9 in mediating this response. We show that a single deficiency in TLR2, TLR4, or TLR9 has only modest, selective effects on cytokine and chemokine secretion, whereas substantial defects were observed in TLR2(-/-)xTLR9(-/-) and TLR2(-/-)xTLR4(-/-) mice, though not as severe as in MyD88(-/-) mice. Chloroquine, which inhibits the function of intracellular TLRs, including TLR9, completely abrogated detectable cytokine and chemokine release in spleen cells from TLR2(-/-)xTLR4(-/-) mice, similar to what is observed for mice deficient in MyD88. These data demonstrate significant synergy between TLR2 and both TLR4 and TLR9 for induction of the MyD88-dependent splenic cytokine and chemokine response to Pn.     

Extracellular histones disarrange vasoactive mediators release through a COX‐NOS interaction in human endothelial cells

Extracellular histones are mediators of inflammation, tissue injury and organ dysfunction. Interactions between circulating histones and vascular endothelial cells are key events in histone‐mediated pathologies. Our aim was to investigate the implication of extracellular histones in the production of the major vasoactive compounds released by human endothelial cells (HUVECs), prostanoids and nitric oxide (NO). HUVEC exposed to increasing concentrations of histones (0.001 to 100 μg/ml) for 4 hrs induced prostacyclin (PGI2) production in a dose‐dependent manner and decreased thromboxane A2 (TXA2) release at 100 μg/ml. Extracellular histones raised cyclooxygenase‐2 (COX‐2) and prostacyclin synthase (PGIS) mRNA and protein expression, decreased COX‐1 mRNA levels and did not change thromboxane A2 synthase (TXAS) expression. Moreover, extracellular histones decreased both, eNOS expression and NO production in HUVEC. The impaired NO production was related to COX‐2 activity and superoxide production since was reversed after celecoxib (10 μmol/l) and tempol (100 μmol/l) treatments, respectively. In conclusion, our findings suggest that extracellular histones stimulate the release of endothelial‐dependent mediators through an up‐regulation in COX‐2‐PGIS‐PGI2 pathway which involves a COX‐2‐dependent superoxide production that decreases the activity of eNOS and the NO production. These effects may contribute to the endothelial cell dysfunction observed in histone‐mediated pathologies.     

Isolation, identification, and characterization of histones from plasmodia of the true slime mold Physarum polycephalum using extraction with guanidine hydrochloride

Histones from plasmodia of the true slime mold Physarum polycephalum have been prepared free of slime by an approach to histone isolation that uses extraction of nuclei with 40% guanidine hydrochloride and chromatography of the extract on Bio-Rex 70. This procedure followed by chromatography or electrophoresis has been used to obtain pure fractions of histones from Physarum microplasmodia. Physarum microplasmodia have five major histone fractions, and we show by amino acid analysis, apparent molecular weight on three gel systems containing sodium dodecyl sulfate, mobility on gels containing Triton X-100, and other characterizations that these fractions are analogous to mammalian histones H1, H2A, H2B, H3, and H4. Significant differences between Physarum and mammalian histones are noted, with histone H1 showing by far the greatest variation. Histones H1 and H4 from Physarum microplasmodia have similar, but not identical, products of partial chymotryptic digestion compared with those of calf thymus histones H1 and H4. Labeling experiments, in vivo, showed that histone H1 is the major phosphorylated histone and approximately 15 separate phosphopeptides are present in a tryptic digest of Physarum histone H1. The core histones from Physarum, histones H2A, H2B, H3, and H4, are rapidly acetylated; histone H4 shows five subfractions, analogous to the five subfractions of mammalian histone H4 (containing zero to four acetyllysine residues per molecule); histone H3 has a more complex pattern that we interpret as zero to four acetyllysine residues on each of two sequence variants of histone H3; histones H2A and H2B show less heterogeneity. Overall, the data show that Physarum microplasmodia have a set of histones that is closely analogous to mammalian histones.     

Extracellular Histones Induce Chemokine Production in Whole Blood Ex Vivo and Leukocyte Recruitment In Vivo

The innate immune system relies to a great deal on the interaction of pattern recognition receptors with pathogen- or damage-associated molecular pattern molecules. Extracellular histones belong to the latter group and their release has been described to contribute to the induction of systemic inflammatory reactions. However, little is known about their functions in the early immune response to an invading pathogen. Here we show that extracellular histones specifically target monocytes in human blood and this evokes the mobilization of the chemotactic chemokines CXCL9 and CXCL10 from these cells. The chemokine induction involves the toll-like receptor 4/myeloid differentiation factor 2 complex on monocytes, and is under the control of interferon-γ. Consequently, subcutaneous challenge with extracellular histones results in elevated levels of CXCL10 in a murine air pouch model and an influx of leukocytes to the site of injection in a TLR4 dependent manner. When analyzing tissue biopsies from patients with necrotizing fasciitis caused by Streptococcus pyogenes, extracellular histone H4 and CXCL10 are immunostained in necrotic, but not healthy tissue. Collectively, these results show for the first time that extracellular histones have an important function as chemoattractants as their local release triggers the recruitment of immune cells to the site of infection.     

Human Myocardium Releases Heat Shock Protein 27 (HSP27) after Global Ischemia: The Proinflammatory Effect of Extracellular HSP27 through Toll-like Receptor (TLR)-2 and TLR4

The myocardial inflammatory response contributes to cardiac functional injury associated with heart surgery obligating global ischemia/reperfusion (I/R). Toll-like receptors (TLRs) play an important role in the mechanism underlying myocardial I/R injury. The aim of this study was to examine the release of small constitutive heat shock proteins (HSPs) from human and mouse myocardium after global ischemia and examine the role of extracellular small HSP in myocardial injury. HSP27 release was assessed by enzyme-linked immunosorbent assay. Anti-HSP27 was applied to evaluate the role of extracellular HSP27 in the postischemic inflammatory response and functional injury in mouse hearts. Isolated hearts and cultured coronary vascular endothelial cells were exposed to recombinant HSP27 to determine its effect on proinflammatory signaling and production of proinflammatory mediators. HSP27 levels were markedly elevated in coronary sinus blood of patients and in coronary effluent of mouse hearts after global ischemia. Neutralizing extracellular HSP27 suppressed myocardial nuclear factor (NF)-κB activation and interleukin (IL)-6 production and improved cardiac function in mouse hearts. Perfusion of HSP27 to mouse hearts induced NF-κB activation and IL-6 production and depressed contractility. Further, recombinant HSP27 induced NF-κB phosphorylation and upregulated monocyte chemoattractant protein (MCP)-1 and intercellular adhesion molecule (ICAM)-1 production in both human and mouse coronary vascular endothelial cells. TLR2 knockout (KO) or TLR4 mutation abolished NF-κB phosphorylation and reduced MCP-1 and ICAM-1 production induced by extracellular HSP27 in endothelial cells. In conclusion, these results show that the myocardium releases HSP27 after global ischemia and that extracellular HSP27 is proinflammatory and contributes to the inflammatory mechanism of myocardial functional injury. Both TLR2 and TLR4 are involved in mediating the proinflammatory effect of extracellular HSP27.     

HISTONES AS EXTRACELLULAR MESSENGERS: EFFECTS ON GROWTH HORMONE SECRETION

Histones display hormone-like properties when present in extracellular fluids. The authors report that histones H2A and H2B possess growth hormone (GH)-releasing activityin vitroand describe the specificity and signal transduction pathways involved in these effects. Perfused and incubated rat pituitary cells were used in different sets of experiments and GH release was measured by radio-immunoassay (RIA). Perfusion of cells with 30μmhistone H2A or H2B, generated significant GH secretory responses. Cells incubated with histone H2A showed a dose- and time-dependent stimulatory effect on GH release which was blocked by peptide MB35, a synthetic fragment of histone H2A. Incubation of pituitary cells with the GH secretagogue GHRP-6, and histones revealed an additive release of GH, whereas GHRH and histones revealed a synergistic effect. The basic peptide poly-Lys did not mimetize the action of histones. Both EGTA and the protein kinase C inhibitor trifluoperazine, but not the calcium ionophre A23187, were able to reduce significantly the GH response of somatotrophs to histones. Pituitary cell incubation with 30μmforskolin alone or in the presence of H2A or H2B, stimulated GH release in the same magnitude. The results confirm previous evidence that histones may act as hypophysotropic signals and suggest, although do not prove, that this activity is receptor dependent. Calcium- and diacylglycerol-associated pathways participate in these effects.     

Neutrophil extracellular traps impair intestinal barrier functions in sepsis by regulating TLR9-mediated endoplasmic reticulum stress pathway

Increased neutrophil extracellular traps (NETs) formation has been found to be associated with intestinal inflammation, and it has been reported that NETs may drive the progression of gut dysregulation in sepsis. However, the biological function and regulation of NETs in sepsis-induced intestinal barrier dysfunction are not yet fully understood. First, we found that both circulating biomarkers of NETs and local NETs infiltration in the intestine were significantly increased and had positive correlations with markers of enterocyte injury in abdominal sepsis patients. Moreover, the levels of local citrullinated histone 3 (Cit H3) expression were associated with the levels of BIP expression. To further confirm the role of NETs in sepsis-induced intestinal injury, we compared peptidylarginine deiminase 4 (PAD4)-deficient mice and wild-type (WT) mice in a lethal septic shock model. In WT mice, the Cit H3-DNA complex was markedly increased, and elevated intestinal inflammation and endoplasmic reticulum (ER) stress activation were also found. Furthermore, PAD4 deficiency alleviated intestinal barrier disruption and decreased ER stress activation. Notably, NETs treatment induced intestinal epithelial monolayer barrier disruption and ER stress activation in a dose-dependent manner in vitro, and ER stress inhibition markedly attenuated intestinal apoptosis and tight junction injury. Finally, TLR9 antagonist administration significantly abrogated NETs-induced intestinal epithelial cell death through ER stress inhibition. Our results indicated that NETs could contribute to sepsis-induced intestinal barrier dysfunction by promoting inflammation and apoptosis. Suppression of the TLR9–ER stress signaling pathway can ameliorate NETs-induced intestinal epithelial cell death.Subject terms: Mucosal immunology, Intestinal diseases, Sepsis     

Reversible effects of Na-butyrate on histone acetylation

Histones were labeled by incubating HeLa cells in the presence of radioactive leucine for 20 hours. Following a 5 hour chase in non-radioactive medium the cells were exposed to 7 mM Na-butyrate to increase the level of histone acetylation. Histones were then extracted, fractionated by high-resolution electrophoresis in acetic acid-urea gels and the specific activity of the parental form of H4 histone and that of each acetylated form was calculated. No differences were found in the specific activities indicating that the major effect of butyrate on histone acetyl levels involves histones which were synthesized before the administration of butyrate. The effect is reversible and within 15 minutes after the removal of the drug most of the acetylated forms of H4 histone are converted to the unmodified form.     

Phosphorylation of rat thymus histones, its control and the effects thereon of gamma-irradiation.

The phosphate content of rat thymus histones was determined. As expected for a replicating tissue, histones 1 and 2B were more phosphorylated and had higher 32P uptakes than did histones from resting liver nuclei; the other histones all showed 32P uptake, but the phosphate content and uptake of histone 2A was about half that for liver histone 2A. When thymus nuclei were incubated in a slightly hypo-osmotic medium, non-histone proteins and phosphorylated histones were released into solution; this was enhanced if ATP was present in the medium. [gamma-32P]ATP was incorporated into non-histone proteins, including protein P1, and into the ADP-ribosylated form of histone 1; negligible 32P was incprporated into the other, bound, histones. Histones 1 and 2B added to the incubation medium were extensively, and histones 2A and 4 slightly, phosphorylated. Histones released by increasing the ionic strength of the medium were phosphorylated. Added lysozyme and cytochrome c were neither bound nor phosphorylated, but added non-histone protein P1 was phosphorylated, causing other histones to be released from the nuclei, especially histones 2A and 3. The released histones were phosphorylated. gamma-Irradiation decreased 32P uptake into the non-ADP-ribosylated histones 1 and 4; phosphorylation of histone 1 in vitro was unaffected. The importance of non-histone proteins, ATP availability and nuclear protein kinases to the control of histone phosphorylation in vivo is discussed.     

Engagement of Toll-like receptors by mycoplasmal superantigen: downregulation of TLR2 by MAM/TLR4 interaction

Mycoplasma arthritidis mitogen (MAM) is a superantigen (SAg) from M. arthritidis, an agent of murine toxic shock syndrome and arthritis. We previously demonstrated that C3H/HeJ and C3H/HeSnJ mice that differ in expression of TLR4 differed in immune reactivity to MAM. We show here that MAM directly interacts with TLR2 and TLR4 by using monoclonal antibodies to TLR2 and TLR4 which inhibit cytokine responses of THP-1 cells to MAM. Also, using macrophages from C3H substrains and TLR2-deficient mice, we confirmed that both TLR2 and TLR4 are used by MAM. Levels of IL-6 in supernatants of MAM-challenged macrophages were higher in mice which expressed only TLR2, lesser with both TLR2 and TLR4, and absent in mice lacking both TLR2 and TLR4. In addition, expression of TLR2 and TLR4 was moderately upregulated in wild-type cells but cells lacking TLR4 showed a fivefold increase in TLR2 expression. Further, blockade of TLR4 on macrophages of C3H/HeN mice with antibody greatly increased expression of TLR2 and release of IL-12p40 in response to MAM. These results indicate that the SAg, MAM, interacts with both TLR2 and TLR4 and that TLR4 signalling might downregulate the MAM/TLR2 inflammatory response.     

Hyaluronic Acid Oligosaccharides Suppress TLR3-Dependent Cytokine Expression in a TLR4-Dependent Manner

The release of endogenous molecules from the skin after injury has been proposed to influence inflammation. Recent studies have found that pro-inflammatory signals can be generated by damaged endogenous self-RNA, and this event is detected by TLR3. Conversely, release of endogenous fragments of hyaluronic acid (HA) after injury has been proposed to inhibit LPS induced inflammation driven by TLR4. In this study we investigated if HA oligomers could also influence inflammation mediated by TLR3. A tetramer form of HA (oligo-HA) was added to MH-S cells (mouse alveolar macrophage cell line) that were then activated by poly(I:C). ELISA analysis of culture supernatants showed that the presence of oligo-HA suppressed the poly(I:C) induced release of IL-6 and TNFα. IL-6 mRNA expression was also suppressed as measured by quantitative RT-PCR. To determine the mechanism of action for oligo-HA to inhibit poly(I:C), macrophages derived from wild-type (WT), Tlr2−/− or Tlr4−/− mice were treated with oligo-HA and poly(I:C). Similar to WT cells, Tlr2−/− macrophages were inhibited by oligo-HA and retained suppression of cytokine release. In contrast, Tlr4−/− macrophages lost the capacity to be suppressed by oligo-HA. An increase in Traf1 (TLR negative regulator) mRNA was observed after oligo-HA treatment of WT but not in Tlr4−/− macrophages, and oligo-HA did not suppress cytokine responsiveness in Traf1−/− macrophages. These results show that oligo-HA acts through TLR4 and TRAF1 to inhibit TLR3-dependent inflammation. This observation illustrates the complex immunomodulatory action of endogenous products released after injury.     

Cutting edge: all-trans retinoic acid down-regulates TLR2 expression and function

A major consequence of microbial infection is the tissue injury that results from the host inflammatory response. In acne, inflammation is due in part to the ability of Propionibacterium acnes to activate TLR2. Because all-trans retinoic acid (ATRA) decreases inflammation in acne, we investigated whether it regulates TLR2 expression and function. Treatment of primary human monocytes with ATRA led to the down-regulation of TLR2 as well as its coreceptor CD14, but not TLR1 or TLR4. The ability of a TLR2/1 ligand to trigger monocyte cytokine release was inhibited by pre- and cotreatment with ATRA; however, TLR4 activation was affected by cotreatment only. ATRA also down-regulated monocyte cytokine induction by P. acnes. These data indicate that ATRA exerts an anti-inflammatory effect on monocytes via two pathways, one specifically affecting TLR2/1 and CD14 expression and one independent of TLR expression. Agents that target TLR expression and function represent a novel strategy to treat inflammation in humans.     

Destabilization of the outer and inner mitochondrial membranes by core and linker histones

Background

Extensive DNA damage leads to apoptosis. Histones play a central role in DNA damage sensing and may mediate signals of genotoxic damage to cytosolic effectors including mitochondria.

Methodology/Principal Findings

We have investigated the effects of histones on mitochondrial function and membrane integrity. We demonstrate that both linker histone H1 and core histones H2A, H2B, H3, and H4 bind strongly to isolated mitochondria. All histones caused a rapid and massive release of the pro-apoptotic intermembrane space proteins cytochrome c and Smac/Diablo, indicating that they permeabilize the outer mitochondrial membrane. In addition, linker histone H1, but not core histones, permeabilized the inner membrane with a collapse of the membrane potential, release of pyridine nucleotides, and mitochondrial fragmentation.

Conclusions

We conclude that histones destabilize the mitochondrial membranes, a mechanism that may convey genotoxic signals to mitochondria and promote apoptosis following DNA damage.     

Sites of in vivo histone methylation in developing trout testis.

Specific lysyl residues of trout testis histones H3 and H4 are methylated partially during rainbow trout spermatogenesis. Histones H1, H2A, H2B, and protamine are not methylated. The single site (lysine 20) in histone H4 and the two major sites (lysines 9 and 27) in histone H3 are homologous to those determined for other organisms, but an additional minor site (lysine 4) occurs in histone H3. As described for calf thymus, both histones H3 and H4 contain epsilon-N-mono- and dimethyllysine, while histone H3 contains in addition, epsilon-N-trimethyllysine. The trout-specific histone H6, which accounts for 0.5 to 1.0% of total histone, contains a sequence for residues 3 to 5,-Arg-Lys-Ser-, which is the same as one methylated in histones H3, at lysines 9 and 27. However, histone H6 yields only trace amounts of [3H]methyl incorporation and no detectable methyllysines on amino acid analysis.     

Importance of extra- and intracellular domains of TLR1 and TLR2 in NFkappa B signaling

Recognition of ligands by toll-like receptor (TLR) 2 requires interactions with other TLRs. TLRs form a combinatorial repertoire to discriminate between the diverse microbial ligands. Diversity results from extracellular and intracellular interactions of different TLRs. This paper demonstrates that TLR1 and TLR2 are required for ara-lipoarabinomannan- and tripalmitoyl cysteinyl lipopeptide-stimulated cytokine secretion from mononuclear cells. Confocal microscopy revealed that TLR1 and TLR2 cotranslationally form heterodimeric complexes on the cell surface and in the cytosol. Simultaneous cross-linking of both receptors resulted in ligand-independent signal transduction. Using chimeric TLRs, we found that expression of the extracellular domains along with simultaneous expression of the intracellular domains of both TLRs was necessary to achieve functional signaling. The domains from each receptor did not need to be contained within a single contiguous protein. Chimeric TLR analysis further defined the toll/IL-1R domains as the area of crucial intracellular TLR1-TLR2 interaction.     

Involvement of toll-like receptor (TLR) 2 and TLR4 in cell activation by mannuronic acid polymers

The alginate capsule produced by the human pathogen Pseudomonas aeruginosa is composed mainly of mannuronic acid polymers (poly-M) that have immunostimulating properties. Poly-M shares with lipopolysaccharide the ability to stimulate cytokine production from human monocytes in a CD14-dependent manner. In the present study we examined the role of Toll-like receptor (TLR) 2 and TLR4 in responses to poly-M. Blocking antibodies to TLR2 and TLR4 partly inhibited tumor necrosis factor production induced by poly-M in human monocytes, and further inhibition was obtained by combining the antibodies. By transiently transfecting HEK293 cells, we found that membrane CD14 together with either TLR2 or TLR4/MD-2 could mediate activation by poly-M. Transfection of HEK293 cells with TLR2 and fluorescently labeled TLR4 followed by co-patching of TLR2 with an antibody revealed no association of these molecules on the plasma membrane. However, macrophages from the Tlr4 mutant C3H/HeJ mice and TLR4 knockout mice were completely non-responsive to poly-M, whereas the tumor necrosis factor release from TLR2 knockout macrophages was half of that seen with wild type cells. Taken together the results suggest that both TLR2 and TLR4 are involved in cell activation by poly-M and that TLR4 may be required in primary murine macrophages.