Neutrophil extracellular traps induced by the hypoxic microenvironment in gastric cancer augment tumour growth.

BACKGROUND: Inflammation-related predisposition to cancer plays an essential role in cancer progression and is associated with poor prognosis. A hypoxic microenvironment and neutrophil infiltration are commonly present in solid tumours, including gastric cancer (GC). Neutrophil extracellular traps (NETs) have also been demonstrated in the tumour immune microenvironment (TIME), but how NETs affect GC progression remains unknown. Here, we investigated the role of NET formation in the TIME and further explored the underlying mechanism of NETs in GC tumour growth. METHODS: Hypoxia-induced factor-1α (HIF-1α), citrulline histone 3 (citH3) and CD66b expression in tumour and adjacent nontumor tissue samples was evaluated by western blotting, immunofluorescence and immunohistochemical staining. The expression of neutrophil-attracting chemokines in GC cells and their hypoxic-CM was measured by qRT‒PCR and ELISA. Neutrophil migration under hypoxic conditions was evaluated by a Transwell assay. Pathway activation in neutrophils in a hypoxic microenvironment were analysed by western blotting. NET formation was measured in vitro by immunofluorescence staining. The protumour effect of NETs on GC cells was identified by Transwell, wound healing and cell proliferation assays. In vivo, an lipopolysaccharide (LPS)-induced NET model and subcutaneous tumour model were established in BALB/c nude mice to explore the mechanism of NETs in tumour growth. RESULTS: GC generates a hypoxic microenvironment that recruits neutrophils and induces NET formation. High mobility group box 1 (HMGB1) was translocated to the cytoplasm from the nucleus of GC cells in the hypoxic microenvironment and mediated the formation of NETs via the toll-like receptor 4 (TLR4)/p38 MAPK signalling pathway in neutrophils. HMGB1/TLR4/p38 MAPK pathway inhibition abrogated hypoxia-induced neutrophil activation and NET formation. NETs directly induced GC cell invasion and migration but not proliferation and accelerated the augmentation of GC growth by increasing angiogenesis. This rapid tumour growth was abolished by treatment with the NET inhibitor deoxyribonuclease I (DNase I) or a p38 MAPK signalling pathway inhibitor. CONCLUSIONS: Hypoxia triggers an inflammatory response and NET formation in the GC TIME to augment tumour growth. Targeting NETs with DNase I or HMGB1/TLR4/p38 MAPK pathway inhibitors is a potential therapeutic strategy to inhibit GC progression. Video Abstract.

Pathogen manipulation of chloroplast function triggers a light-dependent immune recognition.

In plants and animals, nucleotide-binding leucine-rich repeat (NLR) proteins are intracellular immune sensors that recognize and eliminate a wide range of invading pathogens. NLR-mediated immunity is known to be modulated by environmental factors. However, how pathogen recognition by NLRs is influenced by environmental factors such as light remains unclear. Here, we show that the agronomically important NLR Rpi-vnt1.1 requires light to confer disease resistance against races of the Irish potato famine pathogen Phytophthora infestans that secrete the effector protein AVRvnt1. The activation of Rpi-vnt1.1 requires a nuclear-encoded chloroplast protein, glycerate 3-kinase (GLYK), implicated in energy production. The pathogen effector AVRvnt1 binds the full-length chloroplast-targeted GLYK isoform leading to activation of Rpi-vnt1.1. In the dark, Rpi-vnt1.1-mediated resistance is compromised because plants produce a shorter GLYK-lacking the intact chloroplast transit peptide-that is not bound by AVRvnt1. The transition between full-length and shorter plant GLYK transcripts is controlled by a light-dependent alternative promoter selection mechanism. In plants that lack Rpi-vnt1.1, the presence of AVRvnt1 reduces GLYK accumulation in chloroplasts counteracting GLYK contribution to basal immunity. Our findings revealed that pathogen manipulation of chloroplast functions has resulted in a light-dependent immune response.

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats.

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory bowel diseases and diabetes. Here, we describe a protocol for the in vitro characterization of the electrophysiological properties of colonic afferents in rats. The colorectum, with the intact pelvic ganglion (PG) attached, is removed from the rat; superfused with carbogenated Krebs solution in the recording chamber; and cannulated at the oral and anal ends to allow for distension. A fine nerve bundle emanating from the PG is identified, and the multiunit afferent nerve activity is recorded using a suction electrode. Distension of the colonic segment elicits gradual increases in multiunit discharge. A principal component analysis is conducted to differentiate the low-threshold, the high-threshold, and the wide-dynamic range afferent fibers. Chemical sensitivity of colonic afferents can be studied through the bath or intraluminal administration of test compounds. This protocol can be modified for application to other species, such as mice and guinea pigs, and to study the differences in the electrophysiological properties of thoracolumbar/hypogastric and lumbosacral/pelvic afferents of the descending colon in normal and pathological conditions.

Impairments of the primary afferent nerves in a rat model of diabetic visceral hyposensitivity.

BACKGROUND: Diabetic neuropathy in visceral organs such as the gastrointestinal (GI) tract is still poorly understood, despite that GI symptoms are among the most common diabetic complications. The present study was designed to explore the changes in visceral sensitivity and the underlying functional and morphological deficits of the sensory nerves in short-term diabetic rats. Here, we compared the colorectal distension (CRD)-induced visceromotor response (VMR, an index of visceral pain) in vivo, the mechanosensitivity of colonic afferents ex vivo as well as the expression of protein gene product (PGP) 9.5 and calcitonin gene-related peptide (CGRP) in colon between diabetic (3-6 weeks after streptozotocin injection) and control (age-matched vehicle injection) rats. RESULTS: VMR was markedly decreased in the diabetic compared to the control rats. There was a significant decrease in multiunit pelvic afferent nerve responses to ramp distension of the ex vivo colon and single unit analysis indicated that an impaired mechanosensitivity of low-threshold and wide dynamic range fibers may underlie the afferent hyposensitivity in the diabetic colon. Fewer PGP 9.5- or CGRP-immunoreactive fibers and lower protein level of PGP 9.5 were found in the colon of diabetic rats. CONCLUSIONS: These observations revealed the distinctive feature of colonic neuropathy in short-term diabetic rats that is characterized by a diminished sensory innervation and a blunted mechanosensitivity of the remnant sensory nerves.

Hydrogen sulfide activates the carotid body chemoreceptors in cat, rabbit and rat ex vivo preparations.

We and others previously reported experimental evidence suggesting an important role for hydrogen sulfide (H2S) in oxygen sensing in murine carotid body chemoreceptors. More recent data implicated abnormal H2S-mediated chemoreceptor signaling in pathological conditions such as chronic heart failure and hypertension. However, the idea of H2S as a mediator of oxygen-sensing in chemoreceptors has been challenged. In particular, it was shown that exogenous H2S inhibited the release of neurotransmitters (ACh and ATP) from the cat carotid body, raising the possibility that there exists significant species difference in H2S-mediated signaling in chemoreceptors. This study was designed specifically to determine the effect of H2S on chemoreceptors in different species. We conducted multiunit extracellular recordings of the sinus nerve in the ex vivo carotid body preparation taken from the rat, the cat and the rabbit. As observed in the mouse carotid body, H2S donors (NaHS or Na2S) evoked qualitatively similar excitatory responses of the afferent sinus nerves of the species studied here. The excitatory effects of the H2S donors were concentration-dependent and reversible. The sinus nerve responses to H2S donors were prevented by blockade of the transmission between type I cells and the afferent terminals, as was the response to hypoxia. These results demonstrate that exogenous H2S exerts qualitatively similar excitatory effects on chemoreceptor afferents of different species. The role of endogenous H2S-mediated signaling in carotid body function in different species awaits further investigation.

Deletion of Gpr128 results in weight loss and increased intestinal contraction frequency.

AIM: To generate a Gpr128 gene knockout mouse model and to investigate its phenotypes and the biological function of the Gpr128 gene. METHODS: Bacterial artificial chromosome-retrieval methods were used for constructing the targeting vector. Using homologous recombination and microinjection technology, a Gpr128 knockout mouse model on a mixed 129/BL6 background was generated. The mice were genotyped by polymerase chain reaction (PCR) analysis of tail DNA and fed a standard laboratory chow diet. Animals of both sexes were used, and the phenotypes were assessed by histological, biochemical, molecular and physiological analyses. Semi-quantitative reverse transcription-PCR and Northern blotting were used to determine the tissue distribution of Gpr128 mRNA. Beginning at the age of 4 wk, body weights were recorded every 4 wk. Food, feces, blood and organ samples were collected to analyze food consumption, fecal quantity, organ weight and constituents of the blood and plasma. A Trendelenburg preparation was utilized to examine intestinal motility in wild-type (WT) and Gpr128(-/-) mice at the age of 8 and 32 wk. RESULTS: Gpr128 mRNA was highly and exclusively detected in the intestinal tissues. Targeted deletion of Gpr128 in adult mice resulted in reduced body weight gain, and mutant mice exhibited an increased frequency of peristaltic contraction and slow wave potential of the small intestine. The Gpr128(+/+) mice gained more weight on average than the Gpr128(-/-) mice since 24 wk, being 30.81 ± 2.84 g and 25.74 ± 4.50 g, respectively (n = 10, P < 0.01). The frequency of small intestinal peristaltic contraction was increased in Gpr128(-/-) mice. At the age of 8 wk, the frequency of peristalsis with an intraluminal pressure of 3 cmH2O was 6.6 ± 2.3 peristalsis/15 min in Gpr128(-/-) intestine (n = 5) vs 2.6 ± 1.7 peristalsis/15 min in WT intestine (n = 5, P < 0.05). At the age of 32 wk, the frequency of peristaltic contraction with an intraluminal pressure of 2 and 3 cmH2O was 4.6 ± 2.3 and 3.1 ± 0.8 peristalsis/15 min in WT mice (n = 8), whereas in Gpr128(-/-) mice (n = 8) the frequency of contraction was 8.3 ± 3.0 and 7.4 ± 3.1 peristalsis/15 min, respectively (2 cmH2O: P < 0.05 vs WT; 3 cmH2O: P < 0.01 vs WT). The frequency of slow wave potential in Gpr128(-/-) intestine (35.8 ± 4.3, 36.4 ± 4.2 and 37.1 ± 4.8/min with an intraluminal pressure of 1, 2 and 3 cmH2O, n = 8) was also higher than in WT intestine (30.6 ± 4.2, 31.4 ± 3.9 and 31.9 ± 4.5/min, n = 8, P < 0.05). CONCLUSION: We have generated a mouse model with a targeted deletion of Gpr128 and found reduced body weight and increased intestinal contraction frequency in this animal model.

Hyperpolarization-activated cyclic nucleotide-gated cation channel subtypes differentially modulate the excitability of murine small intestinal afferents.

AIM: To assess the role of hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels in regulating the excitability of vagal and spinal gut afferents. METHODS: The mechanosensory response of mesenteric afferent activity was measured in an ex vivo murine jejunum preparation. HCN channel activity was recorded through voltage and current clamp in acutely dissociated dorsal root ganglia (DRG) and nodose ganglia (NG) neurons retrogradely labeled from the small intestine through injection of a fluorescent marker (DiI). The isoforms of HCN channels expressed in DRG and NG neurons were examined by immunohistochemistry. RESULTS: Ramp distension of the small intestine evoked biphasic increases in the afferent nerve activity, reflecting the activation of low- and high-threshold fibers. HCN blocker CsCl (5 mmol/L) preferentially inhibited the responses of low-threshold fibers to distension and showed no significant effects on the high-threshold responses. The effect of CsCl was mimicked by the more selective HCN blocker ZD7288 (10 µmol/L). In 71.4% of DiI labeled DRG neurons (n = 20) and 90.9% of DiI labeled NG neurons (n = 10), an inward current (I(h) current) was evoked by hyperpolarization pulses which was fully eliminated by extracellular CsCl. In neurons expressing I(h) current, a typical "sag" was observed upon injection of hyperpolarizing current pulses in current-clamp recordings. CsCl abolished the sag entirely. In some DiI labeled DRG neurons, the I(h) current was potentiated by 8-Br-cAMP, which had no effect on the I(h) current of DiI labeled NG neurons. Immunohistochemistry revealed differential expression of HCN isoforms in vagal and spinal afferents, and HCN(2) and HCN(3) seemed to be the dominant isoform in DRG and NG, respectively. CONCLUSION: HCNs differentially regulate the excitability of vagal and spinal afferent of murine small intestine.

Duodenal activation of cAMP-dependent protein kinase induces vagal afferent firing and lowers glucose production in rats.

BACKGROUND & AIMS: The duodenum senses nutrients to maintain energy and glucose homeostasis, but little is known about the signaling and neuronal mechanisms involved. We tested whether duodenal activation of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA) is sufficient and necessary for cholecystokinin (CCK) signaling to trigger vagal afferent firing and regulate glucose production. METHODS: In rats, we selectively activated duodenal PKA and evaluated changes in glucose kinetics during the pancreatic (basal insulin) pancreatic clamps and vagal afferent firing. The requirement of duodenal PKA signaling in glucose regulation was evaluated by inhibiting duodenal activation of PKA in the presence of infusion of the intraduodenal PKA agonist (Sp-cAMPS) or CCK1 receptor agonist (CCK-8). We also assessed the involvement of a neuronal network and the metabolic impact of duodenal PKA activation in rats placed on high-fat diets. RESULTS: Intraduodenal infusion of Sp-cAMPS activated duodenal PKA and lowered glucose production, in association with increased vagal afferent firing in control rats. The metabolic and neuronal effects of duodenal Sp-cAMPS were negated by coinfusion with either the PKA inhibitor H89 or Rp-CAMPS. The metabolic effect was also negated by coinfusion with tetracaine, molecular and pharmacologic inhibition of NR1-containing N-methyl-d-aspartate (NMDA) receptors within the dorsal vagal complex, or hepatic vagotomy in rats. Inhibition of duodenal PKA blocked the ability of duodenal CCK-8 to reduce glucose production in control rats, whereas duodenal Sp-cAMPS bypassed duodenal CCK resistance and activated duodenal PKA and lowered glucose production in rats on high-fat diets. CONCLUSIONS: We identified a neural glucoregulatory function of duodenal PKA signaling.

Sensitivity of bronchopulmonary receptors to cold and heat mediated by transient receptor potential cation channel subtypes in an ex vivo rat lung preparation.

Changes in airway temperature can result in respiratory responses such as cough, bronchoconstriction and mucosal secretion after cold exposure and hyperventilation after heat exposure. In the present investigation, we examined the activity of bronchopulmonary receptors in response to activators of thermo-sensitive transient receptor potential (TS-TRP) cation channels using an ex vivo rat lung preparation. Receptive fields in small bronchioles were probed with von Frey hair monofilaments, warm (50°C) or cold (8°C) saline or saline containing TS-TRP agonists. Among 233 fibers tested, 159 (68.2%) responded to heat (50°C). A large proportion of heat-responsive receptors (107/145) were also activated by capsaicin. Heat and capsaicin-evoked responses were both blocked by TRPV1 antagonist, capsazepine. Only 15.3% of airway receptors responded to cold, which was associated with sensitivity to TRPM8 agonist menthol but not to TRPA1 agonist cinnamaldehyde (CA). Moreover, cold-evoked responses was unaffected by TRPA1 antagonist HC-03001. Our observations suggest that TRPV1 and TRPM8 are involved in transducing heat and cold in the lower respiratory tract, respectively.

A crucial role for hydrogen sulfide in oxygen sensing via modulating large conductance calcium-activated potassium channels.

Hydrogen sulfide (H(2)S) is an important signaling molecule produced from L-cysteine by cystathionine beta-synthetase (CBS) or cystathionine gamma-lyase (CSE). Here we examined the role of H(2)S in the oxygen-sensing function of the carotid body chemoreceptors, where the large conductance Ca(2+)-activated potassium channel (BK(Ca)) plays a key role. In the isolated mouse carotid body/sinus nerve preparations, the H(2)S donor, NaHS, excited the chemoreceptor afferent nerves in a concentration-dependent manner that was reversed by carbon monoxide donor. The NaHS-evoked excitation was abolished by removing extracellular Ca(2+), or using Cd(2+), pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid and hexomethonium, suggesting that H(2)S evokes release of ATP/ACh from type I glomus cells of the carotid body. The chemoreceptor afferent activation by hypoxia was decreased remarkably using CBS inhibitors, amino oxyacetic acid (AOAA) and hydroxylamine, but not CSE inhibitors, propargylglycine and beta-cyano-L-alanine, despite expression of both enzymes in type I glomus cells. In these cells, the BK(Ca) currents were inhibited by hypoxia and such inhibition was mimicked by NaHS and diminished by AOAA. Finally, mice hyperventilated in response to hypoxia, which was prevented by CBS inhibitors. These data suggest that H(2)S plays a crucial role in mediating the response of carotid body chemoreceptors to hypoxia via modulating the BK(Ca) channels.

Ion channel and receptor mechanisms of bladder afferent nerve sensitivity.

Sensory nerves of the urinary bladder consist of small diameter A(delta) and C fibers running in the hypogastic and pelvic nerves. Neuroanatomical studies have revealed a complex neuronal network within the bladder wall. Electrophysiological recordings in vitro and in vivo have revealed several distinct classes of afferent fibers that may signal a wide range of bladder stimulations including physiological bladder filling, noxious distension, cold, chemical irritation and inflammation. The exact mechanisms that underline mechanosensory transduction in bladder afferent terminals remain ambiguous; however, a wide range of ion channels (e.g., TTX-resistant Na(+) channels, Kv channels and hyperpolarization-activated cyclic nucleotide-gated cation channels) and receptors (e.g., TRPV1, TRPM8, TRPA1, P2X(2/3), etc) have been identified at bladder afferent terminals and implicated in the generation and modulation of afferent signals. Experimental investigations have revealed that expression and/or function of these ion channels and receptors may be altered in animal models and patients with overactive and painful bladder disorders. Some of these ion channels and receptors may be potential therapeutic targets for bladder diseases.

The role of metabotropic glutamate receptor mGlu5 in control of micturition and bladder nociception.

In micturition control, the roles of ionotropic glutamate (iGlu) receptors NMDA and AMPA are well established, whereas little is known about the function of metabotropic glutamate (mGlu) receptors. Since antagonists for mGlu5 receptors are efficacious in animal models of inflammatory and neuropathic pain, we examined whether mGlu5 receptors play a role in the voiding reflex and bladder nociception and, if so, via centrally or peripherally localized receptors. The mGlu5 receptor antagonist MPEP dose-dependently increased the micturition threshold (MT) volume in the volume-induced micturition reflex (VIMR) model in anesthetized rats. Following doses of 5.2, 15.5 and 51.7micromol/kg of MPEP (intraduodenal), the MT was increased by 24.7+/-5.0%, 97.2+/-12.5% (P<0.01) and 128.0+/-28.3% (P<0.01) from the baseline, respectively (n=4-5; compared with 0.8+/-9.1% in the vehicle group). Infusing MPEP (0.3, 1mM) directly into the bladder also raised MT. However, the efficacious plasma concentrations of MPEP following intravesical dosing were similar to that after intraduodenal dosing (EC(50) of 0.11 and 0.27microM, respectively, P>0.05). MPEP also dose-dependently attenuated the visceromotor responses (VMR, total number of abdominal EMG spikes during phasic bladder distension) in anesthetized rats. The VMR was decreased to 1332.4+/-353.9 from control of 2886.5+/-692.2 spikes/distension (n=6, P<0.01) following MPEP (10micromol/kg, iv). Utilizing the isolated mouse bladder/pelvic nerve preparation, we found that neither MPEP (up to 3microM) nor MTEP (up to 10microM) affected afferent discharge in response to bladder distension (n=4-6). In contrast, MPEP attenuated the responses of the mesenteric nerves to distension of the mouse jejunum in vitro. These data suggest that mGlu5 receptors play facilitatory roles in the processing of afferent input from the urinary bladder, and that central rather than peripheral mGlu5 receptors appear to be responsible.