MIP-2 mRNA Research Articles

A NOVEL OLIGONUCLEOTIDE MRNA MIMIC ATTENUATES HEMORRHAGE-INDUCED ACUTE LUNG INJURY.

Hemorrhagic shock (HS) is accompanied by a pronounced activation of the inflammatory response in which acute lung injury (ALI) is one of the most frequent consequences. Among the pivotal orchestrators of this inflammatory cascade, extracellular cold-inducible RNA-binding protein (eCIRP) emerges as a noteworthy focal point, rendering it as a promising target for the management of inflammation and tissue injury. Recently, we have reported that oligonucleotide poly(A) mRNA mimic termed A 12 selectively binds to the RNA binding region of eCIRP and inhibits eCIRP binding to its receptor TLR4. Furthermore, in vivo administration of eCIRP induces lung injury in healthy mice and that mouse deficient in CIRP showed protection from inflammation-associated lung injury. We hypothesize that A 12 inhibits systemic inflammation and ALI in HS. To test the impacts of A 12 on systemic and lung inflammation, extent of inflammatory cellular infiltration and resultant lung damage were evaluated in a mouse model of HS. Male mice were subjected to controlled hemorrhage with a mean arterial pressure of 30 mm Hg for 90 min and then resuscitated with Ringer's lactate solution containing phosphate-buffered saline (vehicle) or A 12 at a dose of 4 nmol/g body weight (treatment). The infusion volume was twice that of the shed blood. At 4 h after resuscitation, mice were euthanized, and blood and lung tissues were harvested. Blood and tissue markers of inflammation and injury were evaluated. Serum markers of injury (lactate dehydrogenase, alanine transaminase, and blood urea nitrogen) and inflammation (TNF-α, IL-6) were increased after HS and A 12 treatment significantly decreased their levels. A 12 treatment also decreased lung levels of TNF-α, MIP-2, and KC mRNA expressions. Lung histological injury score, neutrophil infiltration (Ly6G staining and myeloperoxidase activity), and lung apoptosis were significantly attenuated after A 12 treatment. Our study suggests that the capacity of A 12 in attenuating HS-induced ALI and may provide novel perspectives in developing efficacious pharmaceutics for improving hemorrhage prognosis.

Oxypurinol protects renal ischemia/reperfusion injury via heme oxygenase-1 induction.

Renal ischemia/reperfusion (I/R) injury is a major cause of acute kidney injury (AKI) by increasing oxidative stress, inflammatory responses, and tubular cell death. Oxypurinol, an active metabolite of allopurinol, is a potent anti-inflammatory and antioxidant agent. To investigate the therapeutic potential and underlying mechanism of oxypurinol in ischemic AKI, C57BL/6 male mice were intraperitoneally injected with oxypurinol and subjected to renal I/R or sham surgery. We found that oxypurinol-treated mice had lower plasma creatinine and blood urea nitrogen levels and tubular damage (hematoxylin-and-eosin staining) compared to vehicle-treated mice after renal I/R injury. Furthermore, oxypurinol treatment reduced kidney inflammation (i.e., neutrophil infiltration and MIP-2 mRNA induction), oxidative stress (i.e., 4-HNE, heme oxygenase-1 [HO-1], 8-OHdG expression, and Catalase mRNA induction), and apoptosis (i.e., TUNEL or cleaved caspase-3-positive renal tubular cells), compared to vehicle-treated mice. Mechanistically, oxypurinol induced protein expressions of HO-1, which is a critical cytoprotective enzyme during ischemic AKI, and oxypurinol-mediated protection against ischemic AKI was completely eliminated by pretreatment with tin protoporphyrin IX, an HO-1 inhibitor. In conclusion, oxypurinol protects against renal I/R injury by reducing oxidative stress, inflammation, and apoptosis via HO-1 induction, suggesting its preventive potential in ischemic AKI.

Hypericum ascyron L. extract reduces particulate matter-induced airway inflammation in mice.

The consequences of increased industrialization increased the risk of asthma and breathing difficulties due to increased particulate matter in the air. We aim to investigate the therapeutic properties of Hypericum ascyron L. extract (HAE) in airway inflammation and unravel its mechanism of action. We conducted nitric oxide and cell viability assay, real-time PCR and western blot analyses along with in vitro studies. in vivo studies include a model of coal fly ash and diesel exhaust particle (CFD)-induced airway inflammation in mice. HAE reduced coal fly ash (CFA)-induced nitric oxide secretion without exhibiting cytotoxicity in MH-S cells. HAE also reduced the mRNA expression of pro-inflammatory cytokines and reduced the expression of proteins in the NFκB and MAPK pathways. In a mice model of CFD-induced airway inflammation, HAE effectively reduced neutrophil infiltration in bronchoalveolar lavage fluid (BALF) and increased the amount of T cells in the BALF, lungs, and blood while reducing all other immune cell subtypes to reduce airway inflammatory response. CXCL-1, IL-17, MIP-2, and TNF-α expression in the BALF were also reduced. HAE effectively reduced MIP-2 and TNF-α mRNA expression in the lung tissue of mice. In a nutshell, HAE is effective in preventing airway inflammation induced by CFA in MH-S cells, as well as inflammation induced by CFD in mice.

Effect of butyl alcohol extract of Baitouweng Decoction on vaginal mucosal neutrophil chemotaxis in vulvovaginal candidiasis mice

To investigate the effects of butyl alcohol extract of Baitouweng Decoction(BAEB) on neutrophil chemotaxis in vaginal mucosa of mice with vulvovaginal candidiasis(VVC). Seventy-two SPF female Kunming mice were randomly divided into normal control group, model group, fluconazole group, BAEB low-dose group, middle-dose group and high-dose group. Subcutaneous injection of estradiol benzoate was conducted to induce pseudo-estrus, and then 2×10~6 CFU·mL~(-1)of Candida albicans was inoculated into vaginal lumen, followed by drug treatment for 7 days. Gram staining was used to observe the morphological changes of C. albicans in vagina; vaginal fungal load was detected on agar plate. Histological changes of vaginal tissues in mice were observed by HE staining. Lactate dehydrogenase(LDH), interleukin-6(IL-6) and tumor necrosis factor(TNF-α) levels in mouse lavage fluid were detected by enzyme-linked immunosorbent assay(ELISA). Neutrophils in vaginal lavage fluid was observed and counted by using Pap smear. The levels of IL-8 and MIP-2 in vaginal mucosa were detected by ELISA. IL-8 and MIP-2 mRNA levels in vaginal mucosa of mice were detected by qRT-PCR. The results showed that as compared with the normal group, VVC model group had a large number of hyphae and a high level of fungal loadinvagina. The vaginal mucosa was completely destroyed, the number of neutrophils increased, and the protein and mRNA levels of IL-8 and MIP-2 were up-regulated. After BAEB treatment, the hyphae of the treatment group was decreased, the fungal load was decreased, the impaired mucosa showed different degrees of improvement, the inflammatory factors were decreased to varying degrees, and the protein and mRNA levels of chemokine IL-8 and MIP-2 were down-regulated. In conclusion, BAEB may be used to treat VVC by inhibiting vulvovaginal candidiasis via blocking neutrophils recruitment into vagina.

Multidrug-resistant Acinetobacter baumannii resists reactive oxygen species and survives in macrophages

We investigated the intracellular survival of multidrug-resistant Acinetobacter baumannii (MDRAB) clinical isolates in macrophages, after phagocytosis, to determine their virulence characteristics. After ATCC 19606 and 5 clinical isolates of MDRAB were phagocytosed by mouse and human macrophages, the bacterial count of MDRAB strains, R4 and R5, increased in the mouse macrophages, 24 hours after phagocytosis. Bacterial count of the strains, R1 and R2, was almost equal 4 and 24 hours after phagocytosis. Intracellular reactive oxygen species was detected in the macrophages after phagocytosis of these bacteria. Further, the strains R1, R2, R4, and R5 showed higher catalase activity than ATCC 19606. Additionally, strains R1, R4, and R5 grew more efficiently than ATCC 19606 in the presence of H2O2, whereas growth of strains R2 and R3 was marginally more than that of ATCC 19606 in the presence of H2O2. The MDRAB clinical isolates altered the expression of TNF-α, IL-1β, IL-6, and MIP-2 mRNA induced in J774A.1 cells, 24 hours after phagocytosis. These results provide insights into the renewed virulence characteristics of MDRAB clinical isolates. Finally, tigecycline killed MDRAB phagocytosed by the macrophages more effectively than colistin, although colistin and tigecycline are both considered effective antibiotics for the treatment of MDRAB.

Role of TFEB in autophagic modulation of ischemia reperfusion injury in mice kidney and protection by urolithin A

Kidney ischemia reperfusion injury (IRI) is an acute kidney injury associated with high number of mortality. We have examined the molecular mechanism and found that oxidative stress and hypoxia leads to induction of autophagy. In IRI induced autophagy, TFEB translocated to nucleus in response to IRI and induced a number of target genes of Coordinated Lysosomal Expression and Regulation (CLEAR) network. Real-time PCR analyses result showed IRI dependent increase in mRNA level to lysosomal hydrolases (Ctsa, Psap), lysosomal membranes (Lamp1), lysosomal acidification (Atp6ap1) non-lysosomal proteins involved in lysosomal biogenesis (M6pr, Nagpa) and autophagy (Becn1, VPS11). Overall, both lysosomal biogenesis and autophagy pathways were induced. Two key players of TFEB dependent proteins in autophagy, LAMP1 and BECN1 were verified by protein analyses. Pretreatment with urolithin A promoted autophagy and attenuated renal injury in kidney IRI and thus inverse relationship existed between TFEB–CLEAR pathway and kidney injury. Urolithin A also attenuated IRI induced pro-inflammatory cytokines TNFα, IL1β, MIP1α and MIP2 mRNA and associated kidney injury. Overall, our results explored the understanding of autophagy and CLEAR network to kidney IRI and those insights may help to develop new therapeutic strategies to protect against IRI.

Abstract 2446: 1-Palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) attenuates gemcitabine-induced neutrophil extravasation

Abstract Neutrophils are the most abundant leukocyte in circulation and are the first responder to foreign particles and/or injury. Some chemotherapeutics are known to induce depletion of blood neutrophils as its high cytotoxicity. However, it is also possible that circulating neutrophils undergo extravasation in response to chemotactic gradients induced by anticancer agents. Here, we found that 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) attenuates gemcitabine-induced neutrophil extravasation via the inhibition of neutrophil-attracting chemokine expression in macrophages using in vivo and in vitro approaches. First, we observed that there were increased neutrophils infiltrated into the peritoneal cavity following gemcitabine treatment in 4T1 tumor-bearing mice. In addition, gemcitabine increased MIP-2 mRNA expression in the peritoneal cells as well as in other tissues of the mice. Likewise, gemcitabine induced the migration of circulating neutrophils into the peritoneal cavity in normal mice, and PLAG effectively decreased neutrophil migration by inhibiting the expression of adhesion molecules. Inhibition of CXCR2 by its antagonist, reparixin, abrogated gemcitabine-induced neutrophil migration, indicating that chemokines produced by gemcitabine mainly support neutrophil activation. In vitro experiments demonstrated that PLAG inhibited NADPH oxidase (NOX)-mediated reactive oxygen species production induced by gemcitabine, which is upstream of MIP-2 and/or CXCL8. Importantly, PLAG downregulated gemcitabine-induced membrane translocation of the cytosolic NOX subunit, Rac1, and phosphorylation of p47phox. We also demonstrated that 1-palmitoyl-2-linoleic-3-hydroxyl-rac-glycerol (PLH), the natural form of diacylglycerol, has no effects on gemcitabine-induced CXCL8 production and dHL-60 migration, suggesting that an acetyl group at the third position of the glycerol backbone may have a key role in the regulation of neutrophil activation. Altogether, this study suggests the potential of PLAG as a therapeutic strategy to modulate chemotherapy-induced neutrophil activation for cancer patients undergoing chemotherapeutic treatment. Citation Format: Jinseon Jeong, Yong Jae Kim, Su-Hyun Shin, Do Young Lee, Byoung-Gon Moon, Ki-Young Sohn, Sun Young Yoon, Jae Wha Kim. 1-Palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) attenuates gemcitabine-induced neutrophil extravasation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2446.

Mincle in the innate immune response of mice fungal keratitis.

To investigate how macrophage inducible C-type lectin (Mincle) influences inflammation in mice fungal keratitis induced by Aspergillus fumigatus (A. fumigatus). C57BL/6 mice were infected with A. fumigatus after pretreated with Mincle agonist TDB or Mincle neutralizing antibody (MincleAb), taking DMSO or IgG as control group respectively. The cornea lesions were monitored with slit-lamp microscope and evaluated by clinical score. Mincle expression was assessed using reverse transcription-ploymerase chain reaction (RT-PCR) and immunostaining. The expression of cytokines (IL-1β, TNF-α and IL-6) chemokines (CXCL-1 and MIP-2) was determined by RT-PCR and ELISA. Neutrophil infiltration was observed by immunostaining. The levels of nitric oxide (NO) generated by corneas were tested by Griess reaction. Mincle mRNA and protein levels were higher in infected corneas than normal corneas of C57BL/6 mice, saving clinical scores revealed differences. When pretreated with Mincle agonist TDB, the mRNA and protein levels of IL-1β, TNF-α and IL-6 in infected corneas were significantly increased compared with the control group (P<0.01). Results of the counterpart in corneas pretreated with Mincle neutralizing antibody was decreased consistently (P<0.01). Expression of CXCL1 and MIP-2 mRNA levels were up-regulated in TDB group and down-regulated in MincleAb group (P<0.01), coincide with neutrophil aggregation degree in corneas showed by immunostaining. As for the concentration of NO, it was promoted in TDB group compared with DMSO control group, and decreased in MincleAb group compared with IgG control group. Mincle plays a dual role in mice fungal keratitis. It participates in the innate immune system by enhancing inflammation. What's more, Mincle can mediate cytotoxic effects by regulating the formation of NO.

Deficiency of receptor-interacting protein kinase 3 (RIPK3) attenuates inflammation and organ injury in neonatal sepsis

IntroductionSepsis is the third leading cause of morbidity and mortality in neonates. Sepsis in neonates is characterized as the systemic inflammation owing to infection within the first 28days after birth. The molecular mechanism causing the exaggerated inflammation phenotype in neonates has not been completely elucidated. Receptor interacting protein kinase 3 (RIPK3) is a protein identified as a mediator in programmed necrosis or necroptosis. We hypothesize that RIPK3 could be responsible for the inflammatory response in neonates and that deficiency in the RIPK3 protein attenuates inflammation and organ injury in neonatal sepsis. MethodsMale and female C57BL6 wild-type (WT) and RIPK3 knock-out (KO) newborn mice aged 5–7days (3–4g body weight) were injected intraperitoneally with 0.9mg/g cecal slurry (CS). At 10h after injection, the newborns were euthanized and blood, the lungs and gut tissues were collected. ResultsAt 10h after CS injection, serum cytokines IL-6 and IL-1β in the WT mice were increased by 511- and 43-fold whereas in KO mice, these levels were increased by 166-fold and 22-fold, respectively. Lung IL-1β in the WT mice increased by 7-fold after CS injection whereas only a 4-fold increase was seen in the KO mice. In the lungs of CS injected KO mice, the injury score, MIP-2 mRNA, myeloperoxidase (MPO) activity and TUNEL staining were significantly reduced by 76%, 70%, 26% and 74%, respectively compared to the CS WT mice. Gut TUNEL staining was also reduced by 80%. ConclusionThe deficiency in RIPK3 attenuated serum and lung cytokines, lung injury and neutrophil infiltration and lung and gut apoptosis. These data suggest that RIPK3, in part, is responsible for the systemic inflammatory response in neonatal sepsis.

New use for an old drug: COX-independent anti-inflammatory effects of sulindac in models of cystic fibrosis.

Background and PurposePulmonary disease is the main cause of morbidity and mortality in cystic fibrosis (CF) patients due to exacerbated inflammation. To date, the only anti‐inflammatory drug available to CF patients is high‐dose ibuprofen, which can slow pulmonary disease progression, but whose cyclooxygenase‐dependent digestive adverse effects limit its clinical use. Here we have tested sulindac, another non‐steroidal anti‐inflammatory drug with an undefined anti‐inflammatory effect in CF airway epithelial cells.Experimental ApproachUsing in vitro and in vivo models, we NF‐κB activity and IL‐8 secretion. In HeLa‐F508del cells, we performed luciferase reporter gene assays in order to measure i) IL‐8 promoter activity, and ii) the activity of synthetic promoter containing NF‐κB responsive elements. We quantified IL‐8 secretion in airway epithelial CFBE cells cultured at an air‐liquid interface and in a mouse model of CF.Key ResultsSulindac inhibited the transcriptional activity of NF‐κB and decreased IL‐8 transcription and secretion in TNF‐α stimulated CF cells via a cyclooxygenase‐independent mechanism. This effect was confirmed in vivo in a mouse model of CF induced by intra‐tracheal instillation of LPS, with a significant decrease of the induction of mRNA for MIP‐2, following treatment with sulindac.Conclusion and ImplicationsOverall, sulindac decrease lung inflammation by a mechanism independent of cycolooxygenase. This drug could be beneficially employed in CF.

MicroRNA Profiling and Regulation in Hypertensive Kidney

Hypertension is a major risk factor for chronic kidney disease and renal inflammation is an integral part in this pathology. In recent years, hydrogen sulfide (H2S) has emerged as an important regulator of blood pressure. Studies in spontaneously hypertensive rats have shown that endogenous hydrogen sulfide (H2S) production is impaired due to decreased CBS/CSE expression, and exogenous H2S supplementation decreased blood pressure and vascular remodeling. MicroRNAs are short, single‐stranded RNA genes that regulate post‐transcriptional gene expression by targeting messenger RNAs (mRNAs), and are associated with a broad spectrum of physiological processes. While there are several studies aimed at investigating the role of non‐coding RNA in hypertension, the roles and implications of these RNAs are not well defined. In this study, we sought to identify microRNAs that are dysregulated in response to angiotensin‐induced hypertension in the kidney and whether hydrogen sulfide donor, GYY 4137, could reverse the microRNA alteration. Wild‐type mice were treated without or with Ang‐II (1000ng/Kg/Min) and without or with GYY4137 for 4 weeks. Blood pressure, renal blood flow, renal resistive index (RI) measurements were performed. microRNA microarrays were conducted and subsequent target prediction revealed genes associated with proinflammatory response. Ang‐II treated mice showed a significant increase in blood pressure accompanied by a decrease in blood flow in the renal cortex and an increase in the resistive index. These effects were attenuated in mice treated with GYY4137. Microarrays of the kidneys revealed downregulation of miR‐129 and miR‐299b in Ang‐II treated mice and upregulation following GYY4137 treatment. In contrast, miR‐369 was upregulated in Ang‐II treatment and suppressed in Ang‐II+GYY4137 mice. Quantitation of genes involved in proinflammatory response revealed upregulation of Tnfα, Il1β, Mcp1 and Mip2 mRNA. Our data suggests that downregulation of miR‐129 and ‐299b plays a significant role in Ang‐II induced renal inflammation, and GYY4137 reduces inflammation and is associated with reversal of miR expression.Support or Funding InformationNIH: HL104103, AHA: 15SDG25840013

1,25-Dihydroxyvitamin D3 attenuates endotoxin-induced production of inflammatory mediators by inhibiting MAPK activation in primary cortical neuron-glia cultures.

BackgroundNeuroinflammation occurs in insulted regions of the brain and may be due to reactive oxygen species (ROS), nitric oxide (NO), cytokines, and chemokines produced by activated glia. Excessive production of neurotoxic molecules causes further neuronal damage. Low levels of vitamin D3 are a risk factor for various brain diseases.MethodsUsing the bacterial endotoxin, lipopolysaccharide (LPS), to induce neuroinflammation in primary cortical neuron-glia cultures, we investigated how 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) affected neuroinflammation.ResultsLPS (100 ng/ml) induced the accumulation of nitrite and the production of ROS, interleukin (IL)-6, and macrophage inflammatory protein (MIP)-2 in time-dependent manners. Inhibition of p38 and extracellular signal-regulated kinase (ERK) but not c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) by 20 μM of SB203580, PD98059, and SP600125, significantly reduced LPS-induced ROS production, NO accumulation, and inducible NO synthase (iNOS) expression, respectively. LPS-induced IL-6 and MIP-2 were significantly attenuated by inhibition of p38, ERK, and JNK MAPK. Cotreatment with 1,25(OH)2D3 attenuated LPS-induced ROS production, NO accumulation, and iNOS expression in concentration-dependent manners. 1,25(OH)2D3 also reduced LPS-induced production of IL-6 and MIP-2. Similarly, iNOS, IL-6, and MIP-2 mRNA expression in cells treated with LPS significantly increased, whereas this effect was attenuated by 1,25(OH)2D3. Moreover, LPS-induced phosphorylation of p38, ERK, and JNK MAPK was significantly inhibited by 1,25(OH)2D3.ConclusionsOur findings indicate that 1,25(OH)2D3 reduced the LPS-stimulated production of inflammatory molecules in neuron-glia cultures by inhibiting MAPK pathways and the production of downstream inflammatory molecules. We suggest that 1,25(OH)2D3 can be used to alleviate neuroinflammation in various brain injuries.

Effect of Different Adjuvants on Protection and Side-Effects Induced by Helicobacter suis Whole-Cell Lysate Vaccination.

Helicobacter suis (H. suis) is a widespread porcine gastric pathogen, which is also of zoonotic importance. The first goal of this study was to investigate the efficacy of several vaccine adjuvants (CpG-DNA, Curdlan, Freund’s Complete and Incomplete, Cholera toxin), administered either subcutaneously or intranasally along with H. suis whole-cell lysate, to protect against subsequent H. suis challenge in a BALB/c infection model. Subcutaneous immunization with Freund’s complete (FC)/lysate and intranasal immunization with Cholera toxin (CT)/lysate were shown to be the best options for vaccination against H. suis, as determined by the amount of colonizing H. suis bacteria in the stomach, although adverse effects such as post-immunization gastritis/pseudo-pyloric metaplasia and increased mortality were observed, respectively. Therefore, we decided to test alternative strategies, including sublingual vaccine administration, to reduce the unwanted side-effects. A CCR4 antagonist that transiently inhibits the migration of regulatory T cells was also included as a new adjuvant in this second study. Results confirmed that immunization with CT (intranasally or sublingually) is among the most effective vaccination protocols, but increased mortality was still observed. In the groups immunized subcutaneously with FC/lysate and CCR4 antagonist/lysate, a significant protection was observed. Compared to the FC/lysate immunized group, gastric pseudo-pyloric metaplasia was less severe or even absent in the CCR4 antagonist/lysate immunized group. In general, an inverse correlation was observed between IFN-γ, IL-4, IL-17, KC, MIP-2 and LIX mRNA expression and H. suis colonization density, whereas lower IL-10 expression levels were observed in partially protected animals.

Effects of intrathecal TRESK gene recombinant adenovirus on inflammatory responses mediated by chemokine in spinal cord of rats with neuropathic pain

Objective To evaluate the effects of intrathecal TRESK gene recombinant adenovirus on inflammatory responses mediated by chemokine in the spinal cord of rats with neuropathic pain (NP) . Methods Thirty-six male Sprague-Dawley rats, weighing 200-250 g, were randomly divided into 6 groups (n=6 each) using a random number table: control group (group C) ; sham operation group (group S) ; NP group; TRESK-overexpressed adenovirus group (group TRESK) ; negative adenovirus group (group Virus) ; normal saline group (group NS) . Spinal nerve injury was produced by exposing the sciatic nerve and its branches and ligation and transection of tibial nerve and common fibular nerve in anesthetized rats.In TRESK, Virus and NS groups, pAd/CMV/V5-DEST-TRESK 25 μl (109 IU/ml) , negative adenovirus 25 μl and normal saline 25 μl were intrathecally injected, respectively.At 1 day before operation (baseline, T0) and 1, 3, 7 and 14 days after operation (T1-4) , the mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency were measured.Six rats in each group were sacrificed after measurement of pain threshold at T3.The L4, 5 segments of the spinal cords were removed for determination of monocyte chemotactic protein-1 (MCP-1) , MIP-2, tumor necrosis factor-alpha (TNF-α) , interleukin-1 beta (IL-1β) and IL-6 mRNA expression by real-time PCR. Results There was no significant difference in thermal paw withdrawal latency at each time point between groups.Compared with C and S groups, MWT at T1-4 in NP and TRESK groups and at T1-3 in Virus and NS groups were significantly decreased, and the expression of MCP-1, MIP-2, TNF-α, IL-1β and IL-6 mRNA was up-regulated in NP, TRESK, Virus and NS groups.Compared with group NP, MWT was significantly increased at T1-4, and the expression of MCP-1, MIP-2, TNF-α, IL-1β and IL-6 mRNA was down-regulated in group TRESK. Conclusion The mechanism by which intrathecal TRESK gene recombinant adenovirus reduces NP is related to inhibition of inflammatory responses mediated by chemokine in the spinal cord of rats. Key words: Potassium channels; Neuralgia; Spinal cord; Receptors, CCR2; Chemokine CXCL2; Inflammatory

Lard-based high-fat diet increases secretory leukocyte protease inhibitor expression and attenuates the inflammatory response of acute lung injury in endotoxemic rats

Acute lung injury (ALI) is less severe in obese than in nonobese patients, but the mechanism is unclear. Secretory leukocyte protease inhibitor (SLPI) is the key anti-inflammatory protein in various lung diseases. We have previously reported changes of the surgical stress in obese rats using lard-based high-fat diet (HFD). The purpose of this study was to elucidate the effect of lard-based HFD on the pathophysiology of lipopolysaccharide (LPS)-induced ALI, and the role of SLPI expression. Male Wistar rats were fed lard-based HFD (60 kcal% fat) or control diet (CD) for either 4 or 12 weeks and were killed after intraperitoneal LPS injection. Analyses included messenger RNA expression of TNF-α, macrophage inflammatory protein (MIP)-2, inducible nitric oxide synthase (iNOS), IL-10 and SLPI in the lung tissue and bronchoalveolar lavage fluid, and histology of the lungs. Rats fed HFD for 12 weeks showed suppression of the lung injury and oxidative stress after LPS injection, as indicated by reduction of pulmonary TNF-α, MIP-2 and iNOS mRNA expression and 8-hydroxy-2'-deoxyguanosine immunostaining. The increased pulmonary SLPI caused by lard was associated with decreased pro-inflammatory cytokines and oxidative stress, which eventually resulted in the prevention of ALI. Those effects of lard on LPS-induced ALI were greater after 12 weeks than after 4 weeks feeding, as indicated by the reduction of TNF-α, MIP-2 and iNOS levels. Feeding lard-based HFD for 12 weeks attenuated LPS-induced ALI with increased pulmonary SLPI expression in rats.

Sa1746 Cationic Amino Acid Transporter 2 and L-Arginine Availability Are Required for Talin-1-Dependent Adherence of the Colonic Pathogen Citrobacter Rodentium and Subsequent Pro-Inflammatory Responses

Background: L-arginine (L-Arg) uptake in cells is mediated by cationic amino acid transporter (CAT) proteins, and CAT2 is the inducible form. We have found that CAT2-/mice exhibit less colitis in the IBD model induced by infection with Citrobacter rodentium, a rodent pathogen similar to EPEC that acts by forming attaching and effacing lesions on epithelial cells. The translocated intimin receptor is injected into host cells by C. rodentium and forms a complex with talin-1 that mediates bacterial binding and downstream signaling. Our aim was to investigate the role of talin-1 in C. rodentium infection. Methods: Male C57BL/6 wild-type (WT) or CAT2-/mice were infected with C. rodentium for 14 days. Talin-1 and the C. rodentium protein espB were assesed by confocal microscopy and Western blotting. mRNA levels of talin-1 and the proinflammatory CXC chemokines, KC and MIP-2, were determined by qPCR. Young adult mouse colon (YAMC) cells were transduced with control or talin-1 shRNA, and activated with C. rodentium at an MOI of 200. Bacterial adherence was assessed by culture after saponin treatment of cells. Levels of the NF-κB subunit p65 were assessed by immunofluorescence at 30 min, and of talin-1, KC and MIP-2 by qPCR at 4 h. Talin-1 mRNA stability was assessed in YAMC cells using the transcriptional inhibitor actinomycin D followed by serial assessment of mRNA levels. Results: C. rodentium infection in vivo induced a 4-fold increase in talin-1 mRNA and protein expression in whole tissues and a 236-fold increase in mRNA levels in colonic epithelial cells (CECs). Talin-1 and espB protein levels were markedly decreased in colon tissues from infected CAT2-/vs. WT mice; this was confirmed by confocal microscopy and there was also less colocalization of these proteins. Talin-1, KC and MIP-2 mRNA levels were each decreased by >92% in CAT2-/vs. WT mice (p<0.01). L-lysine, a competitive inhibitor of L-Arg uptake, reduced induction of talin-1, KC, and MIP-2 mRNA expression by 60.4 ± 18.3%, 91.7 ± 2.6%, and 68.9 ± 12.2%, respectively, in YAMC cells (p<0.01). In talin-1 knockdown cells, levels of adherent C. rodentium decreased from 4.9 ± 0.9 to 0.8 ± 0.2 bacteria/cell (p<0.05), and there was less nuclear translocation of p65. Similarly, KC and MIP-2 levels were decreased by 90.6 ± 0.3% and 92.5 ± 3.1%, respectively, in knockdown cells (p<0.01). There was a 4.2 ± 0.5-fold increase in talin-1 mRNA levels when cells starved of L-Arg were repleted with L-Arg for 4 h. Talin-1 mRNA was less stable in the absence of L-Arg; the mRNA half-life was 5.2fold (p<0.01) greater in cells repleted with L-Arg. Conclusions: Talin-1 is essential for C. rodentium adherence to colonic epithelial cells and induction of host inflammatory responses. L-Arg availability is needed for talin-1 mRNA stability, and thus mediates the immunopathogenesis of C. rodentium infection.

TRAIL-R1 Is a Negative Regulator of Pro-Inflammatory Responses and Modulates Long-Term Sequelae Resulting from Chlamydia trachomatis Infections in Humans

The immune system eliminates Chlamydia trachomatis infection through inflammation. However, uncontrolled inflammation can enhance pathology. In mice, TNF-related apoptosis-inducing ligand receptor (TRAIL-R), known for its effects on apoptosis, also regulates inflammation. In humans, the four homologues of TRAIL-R had never been investigated for effects on inflammation. Here, we examined whether TRAIL-R regulates inflammation during chlamydial infection. We examined TRAIL-R1 single nucleotide polymorphisms (SNPs) in an Ecuadorian cohort with and without C. trachomatis infections. There was a highly significant association for the TRAIL+626 homozygous mutant GG for infection vs no infection in this population. To confirm the results observed in the human population, primary lung fibroblasts and bone marrow-derived macrophages (BMDMs) were isolated from wildtype (WT) and TRAIL-R-deficient mice, and TRAIL-R1 levels in human cervical epithelial cells were depleted by RNA interference. Infection of BMDMs and primary lung fibroblasts with C. trachomatis strain L2, or the murine pathogen C. muridarum, led to higher levels of MIP2 mRNA expression or IL-1β secretion from TRAIL-R-deficient cells than WT cells. Similarly, depletion of TRAIL-R1 expression in human epithelial cells resulted in a higher level of IL-8 mRNA expression and protein secretion during C. trachomatis infection. We conclude that human TRAIL-R1 SNPs and murine TRAIL-R modulate the innate immune response against chlamydial infection. This is the first evidence that human TRAIL-R1 is a negative regulator of inflammation and plays a role in modulating Chlamydia pathogenesis.

Deregulated unfolded protein response in chronic wounds of diabetic ob/ob mice: A potential connection to inflammatory and angiogenic disorders in diabetes-impaired wound healing

Type-2 diabetes mellitus (T2D) represents an important metabolic disorder, firmly connected to obesity and low level of chronic inflammation caused by deregulation of fat metabolism. The convergence of chronic inflammatory signals and nutrient overloading at the endoplasmic reticulum (ER) leads to activation of ER-specific stress responses, the unfolded protein response (UPR). As obesity and T2D are often associated with impaired wound healing, we investigated the role of UPR in the pathologic of diabetic-impaired cutaneuos wound healing. We determined the expression patterns of the three UPR branches during normal and diabetes-impaired skin repair. In healthy and diabetic mice, injury led to a strong induction of BiP (BiP/Grp78), C/EBP homologous protein (CHOP) and splicing of X-box-binding protein (XBP)1. Diabetic-impaired wounds showed gross and sustained induction of UPR associated with increased expression of the pro-inflammatory chemokine macrophage inflammatory protein (MIP)2 as compared to normal healing wounds. In vitro, treatment of RAW264.7 macrophages with tunicamycin, and subsequently stimulation with lipopolysaccharide (LPS) and interferon (IFN)-γ enhances MIP2 mRNA und protein expression compared to proinflammatory stimulation alone. However, LPS/IFNγ induced vascular endothelial growth factor (VEGF) production was blunted by tunicamycin induced-ER stress.Hence, UPR is activated following skin injury, and functionally connected to the production of proinflammatory mediators. In addition, prolongation of UPR in diabetic non-healing wounds aggravates ER stress and weakens the angiogenic phenotype of wound macrophages.

Intranasally Administered Antigen 85B Gene Vaccine in Non-Replicating Human Parainfluenza Type 2 Virus Vector Ameliorates Mouse Atopic Dermatitis

Atopic dermatitis (AD) is a refractory and recurrent inflammatory skin disease. Various factors including heredity, environmental agent, innate and acquired immunity, and skin barrier function participate in the pathogenesis of AD. T -helper (Th) 2-dominant immunological milieu has been suggested in the acute phase of AD. Antigen 85B (Ag85B) is a 30-kDa secretory protein well conserved in Mycobacterium species. Ag85B has strong Th1-type cytokine inducing activity, and is expected to ameliorate Th2 condition in allergic disease. To perform Ag85B function in vivo, effective and less invasive vaccination method is required. Recently, we have established a novel functional virus vector; recombinant human parainfluenza type 2 virus vector (rhPIV2): highly expressive, replication-deficient, and very low-pathogenic vector. In this study, we investigated the efficacy of rhPIV2 engineered to express Ag85B (rhPIV2/Ag85B) in a mouse AD model induced by repeated oxazolone (OX) challenge. Ear swelling, dermal cell infiltrations and serum IgE level were significantly suppressed in the rhPIV2/Ag85B treated mouse group accompanied with elevated IFN-γ and IL-10 mRNA expressions, and suppressed IL-4, TNF-α and MIP-2 mRNA expressions. The treated mice showed no clinical symptom of croup or systemic adverse reactions. The respiratory tract epithelium captured rhPIV2 effectively without remarkable cytotoxic effects. These results suggested that rhPIV2/Ag85B might be a potent therapeutic tool to control allergic disorders.

Age-related changes in phagocytic activity and production of pro-inflammatory cytokines by lipopolysaccharide stimulated porcine alveolar macrophages

The aim of the present study was to determine the age-related changes of phagocytic capacity and the kinetic production of cytokines in lipopolysaccharide-stimulated porcine alveolar macrophages. For this purpose, AMs were isolated from 5 (newborn), 40 (post-weaned) and 120 (young) day old pigs. Results of phagocytosis assay showed that AMs from newborn piglets had less phagocytic capacity than those of young pigs (P<0.05). For the kinetics study, cells and supernatant were collected at 1, 6, 12, 24, 36 and 48h after LPS stimulation for quantification of cytokine mRNA and protein by quantitative real-time PCR and ELISA, respectively. The kinetics results showed that AMs from newborn piglets were significantly less capable of producing IL1β, IL6, IL12β, TNFα and IL8 than post-weaned piglets or young pigs. IL18 mRNA did not show significant differences between ages. MIP2 and MCP1 mRNA was higher in young pigs. Hence, higher production of cytokines by AMs may be the surfactant factors in the pulmonary host defense system. These results indicate that AMs from newborn piglets might be functionally immature, which may lead to increased susceptibility to lung infections. Future studies of cytokine kinetics in more animals are clearly needed to confirm these results across a wider age range.