The Transcription Factor IRF3 Triggers “Defensive Suicide” Necrosis in Response to Viral and Bacterial Pathogens

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Sodium <Emphasis Type=Italic>P</Emphasis>-Aminosalicylic Acid Improved Manganese-Induced Learning and Memory Dysfunction via Restoring the Ultrastructural Alterations and γ-Aminobutyric Acid Metabolism Imbalance in the Basal Ganglia

Excessive intake of manganese (Mn) may cause neurotoxicity. Sodium para-aminosalicylic acid (PAS-Na) has been used successfully in the treatment of Mn-induced neurotoxicity. The γ-aminobutyric acid (GABA) is related with learning and memory abilities. However, the mechanism of PAS-Na on improving Mn-induced behavioral deficits is unclear. The current study was aimed to investigate the effects of PAS-Na on Mn-induced behavioral deficits and the involvement of ultrastructural alterations and γ-aminobutyric acid (GABA) metabolism in the basal ganglia of rats. Sprague-Dawley rats received daily intraperitoneally injections of 15 mg/kg MnCl₂.4H₂O, 5d/week for 4 weeks, followed by a daily back subcutaneously (sc.) dose of PAS-Na (100 and 200 mg/kg), 5 days/week for another 3 or 6 weeks. Mn exposure for 4 weeks and then ceased Mn exposure for 3 or 6 weeks impaired spatial learning and memory abilities, and these effects were long-lasting. Moreover, Mn exposure caused ultrastructural alterations in the basal ganglia expressed as swollen neuronal with increasing the electron density in the protrusions structure and fuzzed the interval of neuropil, together with swollen, focal hyperplasia, and hypertrophy of astrocytes. Additionally, the results also indicated that Mn exposure increased Glu/GABA values as by feedback loops controlling GAT-1, GABA_A mRNA and GABA_A protein expression through decreasing GABA transporter 1(GAT-1) and GABA A receptor (GABA_A) mRNA expression, and increasing GABA_A protein expression in the basal ganglia. But Mn exposure had no effects on GAT-1 protein expression. PAS-Na treatment for 3 or 6 weeks effectively restored the above-mentioned adverse effects induced by Mn. In conclusion, these findings suggest the involvement of GABA metabolism and ultrastructural alterations of basal ganglia in PAS-Na’s protective effects on the spatial learning and memory abilities.     

<Emphasis Type="Italic">Cynomorium songaricum</Emphasis> Extracts Functionally Modulate Transporters of γ-Aminobutyric Acid and Monoamine

Cynomorium songaricum Rupr. (SY) is a central nervous system-oriented herb material that has actions of anti-dementia, anti-epilepsy, and anti-stress. It is unclear whether SY would be biologically active in functionally regulating neurotransmitter transporters. Here, we assessed these potential actions using Chinese hamster ovary cells expressing γ-aminobutyric acid (GABA) transporter (GAT-1), dopamine transporter (DAT), norepinephrine transporter (NET), or serotonin transporter (SERT) (i.e. G1, D8, N1, or S6 cells, respectively). It was shown that SY extracts, such as SYw, SYa, SYp, SYc, SYe, and SYb (SY water, ethanol, petroleum ether, chloroform, ethyl acetate, and n-butyl alcohol extract, respectively) increased dopamine/norepinephrine (DA/NE) uptake by corresponding D8/N1 cells and decreased γ-aminobutyric acid/serotoin (GABA/5HT) uptake by corresponding G1/S6 cells; wherein, the potency or efficacy of SYc for up-regulating DA/NE uptake and that of SYb for inhibiting GABA/5HT uptake were relatively stronger. Additionally, GABA/5HT-uptake inhibition by SY extracts were also seen in cortical synaptosomes, and DA/NE-uptake enhancement by SYc was dependent on the activity of DAT and NET. Thus, SY extracts especially SYc and SYb are novel neurotransmitter-transporter modulators functioning as DAT/NET activators and/or GAT-1/SERT inhibitors.     

Deletion of the γ-Aminobutyric Acid Transporter 2 (GAT2 and SLC6A13) Gene in Mice Leads to Changes in Liver and Brain Taurine Contents

The GABA transporters (GAT1, GAT2, GAT3, and BGT1) have mostly been discussed in relation to their potential roles in controlling the action of transmitter GABA in the nervous system. We have generated the first mice lacking the GAT2 (slc6a13) gene. Deletion of GAT2 (both mRNA and protein) neither affected growth, fertility, nor life span under nonchallenging rearing conditions. Immunocytochemistry showed that the GAT2 protein was predominantly expressed in the plasma membranes of periportal hepatocytes and in the basolateral membranes of proximal tubules in the renal cortex. This was validated by processing tissue from wild-type and knockout mice in parallel. Deletion of GAT2 reduced liver taurine levels by 50%, without affecting the expression of the taurine transporter TAUT. These results suggest an important role for GAT2 in taurine uptake from portal blood into liver. In support of this notion, GAT2-transfected HEK293 cells transported [³H]taurine. Furthermore, most of the uptake of [³H]GABA by cultured rat hepatocytes was due to GAT2, and this uptake was inhibited by taurine. GAT2 was not detected in brain parenchyma proper, excluding a role in GABA inactivation. It was, however, expressed in the leptomeninges and in a subpopulation of brain blood vessels. Deletion of GAT2 increased brain taurine levels by 20%, suggesting a taurine-exporting role for GAT2 in the brain.     

Expression of Rice Glutamate Decarboxylase in <Emphasis Type="Italic">Bifidobacterium Longum</Emphasis> Enhances γ-Aminobutyric Acid Production

Bifidobacteria are important for the production of fermented dairy products and probiotic formulas but have a low capacity for γ-aminobutyric acid (GABA) production. To develop a Bifidobacterium strain with an enhanced GABA production, we transformed Bifidobacterium longum with a rice glutamate decarboxylase (OsGADC⁻) gene by electroporation. When the transformed strain was cultured in medium containing monosodium glutamate, the amount of GABA increased significantly compared with those of untransformed Bifidobacterium. Thus, by introducing a plant derived GAD gene, a Bifidobacterium strain has been genetically engineered to produce high levels of GABA from glutamate.     

IgG Subclass and Heavy Chain Domains Contribute to Binding and Protection by mAbs to the Poly γ-D-glutamic Acid Capsular Antigen of Bacillus anthracis

Bacterial capsules are common targets for antibody-mediated immunity. The capsule of Bacillus anthracis is unusual among capsules because it is composed of a polymer of poly-γ-d-glutamic acid (γdPGA). We previously generated murine IgG3 monoclonal antibodies (mAbs) to γdPGA that were protective in a murine model of pulmonary anthrax. IgG3 antibodies are characteristic of the murine response to polysaccharide antigens. The goal of the present study was to produce subclass switch variants of the γdPGA mAbs (IgG3→IgG1→IgG2b→IgG2a) and assess the contribution of subclass to antibody affinity and protection. Subclass switch antibodies had identical variable regions but differed in their heavy chains. The results showed that a switch from the protective IgG3 to IgG1, IgG2b or IgG2a was accompanied by i) a loss of protective activity ii) a change in mAb binding to the capsular matrix, and iii) a loss of affinity. These results identify a role for the heavy chain constant region in mAb binding. Hybrid mAbs were constructed in which the CH1, CH2 or CH3 heavy chain constant domains from a non-protective, low binding IgG2b mAb were swapped into the protective IgG3 mAb. The IgG3 mAb that contained the CH1 domain from IgG2b showed no loss of affinity or protection. In contrast, swapping the CH2 or CH3 domains from IgG2b into IgG3 produced a reduction in affinity and a loss of protection. These studies identify a role for the constant region of IgG heavy chains in affinity and protection against an encapsulated bacterial pathogen.     

NLRP3/Cryopyrin Is Necessary for Interleukin-1�� (IL-1��) Release in Response to Hyaluronan, an Endogenous Trigger of Inflammation in Response to Injury

Inflammation under sterile conditions is a key event in autoimmunity and following trauma. Hyaluronan, a glycosaminoglycan released from the extracellular matrix after injury, acts as an endogenous signal of trauma and can trigger chemokine release in injured tissue. Here, we investigated whether NLRP3/cryopyrin, a component of the inflammasome, participates in the inflammatory response to injury or the cytokine response to hyaluronan. Mice with a targeted deletion in cryopyrin showed a normal increase in Cxcl2 in response to sterile injuries but had decreased inflammation and release of interleukin-1β (IL-1β). Similarly, the addition of hyaluronan to macrophages derived from cryopyrin-deficient mice increased release of Cxcl2 but did not increase IL-1β release. To define the mechanism of hyaluronan-mediated activation of cryopyrin, elements of the hyaluronan recognition process were studied in detail. IL-1β release was inhibited in peritoneal macrophages derived from CD44-deficient mice, in an MH-S macrophage cell line treated with antibodies to CD44, or by inhibitors of lysosome function. The requirement for CD44 binding and hyaluronan internalization could be bypassed by intracellular administration of hyaluronan oligosaccharides (10–18-mer) in lipopolysaccharide-primed macrophages. Therefore, the action of CD44 and subsequent hyaluronan catabolism trigger the intracellular cryopyrin → IL-1β pathway. These findings support the hypothesis that hyaluronan works through IL-1β and the cryopyrin system to signal sterile inflammation.Inflammation, as defined by changes in vascular permeability and leukocyte recruitment, is an essential step for the control of microbial invasion. Specific microbial products trigger this process through a diverse array of innate immune pattern recognition receptors. However, an inflammatory response independent of infection is also an important process for maintenance of biological homeostasis. For example, normal wound healing requires a controlled inflammatory response to enable the recruitment of monocytes and the release of growth factors required for repair. This response can occur in the absence of microbial stimuli. Furthermore, inflammation and the release of proinflammatory mediators is also associated with many diseases such as rheumatoid arthritis and Crohn disease (1). These diseases are not well understood in terms of their triggers but rather are described by the subsequent release of proinflammatory mediators. Identification of the triggers of sterile inflammation represents an important goal with immediate diagnostic and therapeutic significance.Recent work has begun to elucidate pathways of inflammation that occur in the absence of microbial stimuli. Stress signals such as heat-shock proteins, intracellular components of necrotic cells not normally seen by immune cells, and components of the extracellular matrix have all been implicated as endogenous triggers of injury (2–4). Among this group is the glycosaminoglycan hyaluronan (HA),⁶ an important structural component of the extracellular matrix that is also a common component of bacterial surfaces. HA is synthesized at the cell surface and typically exists as a high molecular mass polymer greater than 10⁶ Da and composed of repeating disaccharide units of N-acetylglucosamine and glucuronic acid (5, 6). Unlike other glycosaminoglycans such as heparan sulfate or chondroitin sulfates that encode specific activity by use of a diverse disaccharide sequence, HA is not sulfated or epimerized, and only changes in HA size, concentration, and location affect function.We have previously developed murine models of sterile injury to identify the innate elements that recognize and mediate sterile inflammation (7). Our results demonstrated that (a) the initiation of a sterile intrinsic inflammatory process is dependent on TLR4 activation, (b) sterile injury induces HA accumulation at the injured site, and (c) sterile intrinsic inflammation resembles signaling events that are activated by HA. Furthermore, we have defined a novel alternative recognition complex for HA that involves TLR4, MD-2, and CD44 (7). Taken together with other work associating HA and innate pattern recognition (4, 8–10), these observations have provided new insight into mechanisms responsible for sterile inflammation.Recently, the NLR (nucleotide-binding domain and leucine rich repeat-containing) family has been extensively analyzed as a group of intracellular pattern recognition receptors (11). NLRs have a leucine-rich repeat that recognizes pathogen-associated molecular patterns including bacterial cell wall components and viral nucleic acids. NOD2 and NLR family, pyrin containing 3 (NLRP3)/cryopyrin are two of the best characterized NLRs. NOD2 recognizes the bacterial peptidoglycan-derived molecule muramyl dipeptide and activates the NF-κB pathway to induce inflammatory responses (12). Mutations of the NOD2 gene were identified in individuals with chronic inflammatory disorders such as Crohn disease (13, 14) and Blau syndrome (15). Mouse knockin mutants of NOD2, which have the same mutation in NOD2 as human patients with Crohn disease, showed elevated proinflammatory cytokines following muramyl dipeptide challenge or dextran sodium sulfate-induced bowel inflammation (16). NLRP3, also known as cyropyrin, CIAS1, NALP3, PYPAF1, forms an “inflammasome” with ASC (apoptosis-associated speck-like protein containing a CARD) and caspase-1 to convert pro-IL-1β to active IL-1β (17). Mutations in NLRP3 were identified in individuals with familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome, and neonatal onset multisystem inflammatory disease (18–20). These individuals have recurrent or chronic inflammatory symptoms, including fever, arthritis, and a urticaria-like eruption characterized by neutrophilic infiltration. In FCAS, symptoms can be elicited by cold provocation by a mechanism that appears to be mediated through the skin (15, 21).Because disorders associated with mutations in NLRP3 are examples of inflammation under sterile conditions and HA has been shown to be a trigger of sterile inflammation, we sought to further understand the mechanism of the response to HA by examining the role of cryopyrin during injury and after exposure to HA. Our results show that cryopyrin and IL-1β are integral to sterile inflammation and the response to HA. These observations provide new insight into the function of HA as a “danger signal” of injury.     

DISC1 Protein Regulates γ-Aminobutyric Acid,Type A (GABAA) Receptor Trafficking and Inhibitory Synaptic Transmission in Cortical Neurons

Association studies have suggested that Disrupted-in-Schizophrenia 1 (DISC1) confers a genetic risk at the level of endophenotypes that underlies many major mental disorders. Despite the progress in understanding the significance of DISC1 at neural development, the mechanisms underlying DISC1 regulation of synaptic functions remain elusive. Because alterations in the cortical GABA system have been strongly linked to the pathophysiology of schizophrenia, one potential target of DISC1 that is critically involved in the regulation of cognition and emotion is the GABA_A receptor (GABA_AR). We found that cellular knockdown of DISC1 significantly reduced GABA_AR-mediated synaptic and whole-cell current, whereas overexpression of wild-type DISC1, but not the C-terminal-truncated DISC1 (a schizophrenia-related mutant), significantly increased GABA_AR currents in pyramidal neurons of the prefrontal cortex. These effects were accompanied by DISC1-induced changes in surface GABA_AR expression. Moreover, the regulation of GABA_ARs by DISC1 knockdown or overexpression depends on the microtubule motor protein kinesin 1 (KIF5). Our results suggest that DISC1 exerts an important effect on GABAergic inhibitory transmission by regulating KIF5/microtubule-based GABA_AR trafficking in the cortex. The knowledge gained from this study would shed light on how DISC1 and the GABA system are linked mechanistically and how their interactions are critical for maintaining a normal mental state.     

Ultra- and Mesostructural Response to Salinization in Two Populations of С3–С4 Intermediate Species Sedobassia sedoides

Russian Journal of Plant Physiology - The plants from two populations (P1 and P2) of xero-halophyte Sedobassia sedoides (Pall.) Freitag &amp; G. Kadereit (Chenopodiaceae) with...     

In Vivo IFN-γ Secretion by NK Cells in Response to Salmonella Typhimurium Requires NLRC4 Inflammasomes

Natural killer (NK) cells are a critical part of the innate immune defense against viral infections and for the control of tumors. Much less is known about how NK cells contribute to anti-bacterial immunity. NK cell-produced interferon gamma (IFN-γ) contributes to the control of early exponential replication of bacterial pathogens, however the regulation of these events remains poorly resolved. Using a mouse model of invasive Salmonellosis, here we report that the activation of the intracellular danger sensor NLRC4 by Salmonella-derived flagellin within CD11c⁺ cells regulates early IFN-γ secretion by NK cells through the provision of interleukin 18 (IL-18), independently of Toll-like receptor (TLR)-signaling. Although IL18-signalling deficient NK cells improved host protection during S. Typhimurium infection, this increased resistance was inferior to that provided by wild-type NK cells. These findings suggest that although NLRC4 inflammasome-driven secretion of IL18 serves as a potent activator of NK cell mediated IFN-γ secretion, IL18-independent NK cell-mediated mechanisms of IFN-γ secretion contribute to in vivo control of Salmonella replication.