MIP-2 Concentrations Research Articles

Experimental Arthritis Inhibits Adult Hippocampal Neurogenesis in Mice.

Background: Adult-born neurons of the hippocampal dentate gyrus play a role in specific forms of learning, and disturbed neurogenesis seems to contribute to the development of neuropsychiatric disorders, such as major depression. Neuroinflammation inhibits adult neurogenesis, but the effect of peripheral inflammation on this form of neuroplasticity is ambiguous. Objective: Our aim was to investigate the influence of acute and chronic experimental arthritis on adult hippocampal neurogenesis and to elucidate putative regulatory mechanisms. Methods: Arthritis was triggered by subcutaneous injection of complete Freund’s adjuvant (CFA) into the hind paws of adult male mice. The animals were killed either seven days (acute inflammation) or 21 days (chronic inflammation) after the CFA injection. Behavioral tests were used to demonstrate arthritis-related hypersensitivity to painful stimuli. We used in vivo bioluminescence imaging to verify local inflammation. The systemic inflammatory response was assessed by complete blood cell counts and by measurement of the cytokine/chemokine concentrations of TNF-α, IL-1α, IL-4, IL-6, IL-10, KC and MIP-2 in the inflamed hind limbs, peripheral blood and hippocampus to characterize the inflammatory responses in the periphery and in the brain. In the hippocampal dentate gyrus, the total number of newborn neurons was determined with quantitative immunohistochemistry visualizing BrdU- and doublecortin-positive cells. Microglial activation in the dentate gyrus was determined by quantifying the density of Iba1- and CD68-positive cells. Results: Both acute and chronic arthritis resulted in paw edema, mechanical and thermal hyperalgesia. We found phagocytic infiltration and increased levels of TNF-α, IL-4, IL-6, KC and MIP-2 in the inflamed hind paws. Circulating neutrophil granulocytes and IL-6 levels increased in the blood solely during the acute phase. In the dentate gyrus, chronic arthritis reduced the number of doublecortin-positive cells, and we found increased density of CD68-positive macrophages/microglia in both the acute and chronic phases. Cytokine levels, however, were not altered in the hippocampus. Conclusions: Our data suggest that acute peripheral inflammation initiates a cascade of molecular and cellular changes that eventually leads to reduced adult hippocampal neurogenesis, which was detectable only in the chronic inflammatory phase.

The Clinical-Stage sGC Stimulator IW-1701 Prevents Increase of Plasma Biomarkers of Intravascular Inflammation and Suppresses Leukocyte-Endothelial Interactions in Tnfα-Treated Mice

Soluble guanylate cyclase (sGC) is a heme containing signaling enzyme that catalyzes the formation of cyclic guanosine 3‘,5‘-monophosphate (cGMP) from GTP in response to nitric oxide (NO) binding to heme. The second messenger cGMP modulates many physiological processes including inflammation. Treatment with NO donors or inhibitors of cGMP-selective phosphodiesterase 9 (PDE9) significantly attenuates TNFα-induced intravascular inflammation in C57BL/6 and sickle cell mice. The anti-inflammatory effect of PDE9 inhibitors was augmented by co-administration of hydroxyurea (HU), which is the current standard of care for sickle cell disease. It has been proposed that HU may exert its anti-inflammatory effect by acting as an NO donor. sGC stimulators are small molecules that bind to sGC and enhance NO-dependent catalytic activity of the enzyme. Here we evaluated the effect of the sGC stimulator IW-1701 in the absence and presence of HU on biomarkers of intravascular inflammation, leukocyte-endothelial cell interactions and neutrophil trafficking in C57BL/6 mice.Treatment of C57BL/6 mice with TNFα (50 ng/mouse, i.p.) increased the plasma levels of biomarkers of endothelial cell activation (sP-selectin, sE-selectin, sICAM-1) and leukocyte activation (MIP-2, sL-selectin). Pretreatment of mice with IW-1701 (10 mg/kg, p.o.) reduced the increase in plasma concentrations of MIP-2 and sL-selectin following the TNFα challenge by 81% and 58%, respectively. The TNFα-induced increase in plasma levels of sP-selectin, sE-selectin and sICAM-1 were also inhibited by 31%, 37% and 34%, respectively in IW-1701-treated mice. Co-treatment with HU augmented the effect of IW-1701 on plasma concentrations of sE-selectin and sICAM-1 in TNFα treated mice.The effects of IW-1701, HU and the combination on leukocyte endothelial cell interactions was assessed using intravital microscopy of post-capillary venules of the mouse cremaster muscle. TNFα treatment of C57BL/6 mice decreased leukocyte velocity from 26.6±3.1 μm/sec to 5.5±0.7 μm/sec and leukocyte rolling flux from 43.3±7.1 cells/min to 16.1±2.7 cells/min as compared to control mice. Pre-treatment of mice with IW-1701 (10 mg/kg) alone increased leukocyte velocity and rolling flux to 10.3±1.1 μm/sec and 24.1±2.3 cells/min, respectively. Similarly, pre-treatment of mice with HU (100 mg/kg, p.o.) alone increased leukocyte velocity to 15.5±1.7 μm/sec and leukocyte rolling flux to 50.2±5.9 cells/min. Co-administration of IW-1701 in combination with HU further increased leukocyte velocity (19.7±1.9 μm/sec) and leukocyte rolling flux (64.5±5.5 cells/min) in post-capillary venules of the mouse cremaster muscle. Treatment with either IW-1701, HU, or the combination did not change the number of adherent leukocytes to the vascular wall following TNFα stimulation. To further evaluate the effect of IW-1701 on neutrophil extravasation and trafficking we used a peritoneal recruitment model. Intraperitoneal injection of TNFα induced accumulation of Gr.1 positive neutrophils in the mouse peritoneal cavity (4±0.4×105 PMNs/peritoneal lavage). Trafficking of PMNs into mouse peritoneum was attenuated by treatment of mice with HU (2.7±0.5×105 PMNs/peritoneal lavage). Co-administration of HU and IW-1701 further decreased the number of neutrophils extravasated into the peritoneum of TNFα-challenged mice (1.4±0.3×105 PMNs/peritoneal lavage).In summary, prophylactic treatment with IW-1701 inhibited the TNFα-induced increase in plasma levels of biomarkers of intravascular inflammation and attenuated the effect of TNFα on transient adhesive interactions between leukocytes and endothelial cells in mice. Co-administration of HU significantly reduced neutrophil trafficking and augmented the effect of IW-1701 on biomarkers of endothelial cell activation and transient adhesive interactions in the mouse cremaster muscle. This study supports further evaluation of the sGC stimulator IW-1701 to reduce complications associated with intravascular inflammation such as vaso-occlusive crisis in patients with sickle cell disease. DisclosuresTchernychev:Ironwood Pharmaceuticals: Employment, Other: own stock options. Feil:Ironwood Pharmaceuticals: Other: This research was sponsored by Ironwood Pharmaceuticals. Germano:Ironwood Pharmaceuticals: Employment, Other: own stock options of Ironwood Pharmaceuticals . Warren:Ironwood Pharmaceuticals: Employment, Other: own stock options of Ironwood Pharmaceuticals . Lonie:Ironwood Pharmaceuticals: Employment, Other: own stock options of Ironwood Pharmaceuticals . Feil:Ironwood Pharmaceuticals: Other: This research was sponsored by Ironwood Pharmaceuticals. Milne:Ironwood Pharmaceuticals: Employment, Equity Ownership. Hadcock:Ironwood Pharmaceuticals: Employment, Other: own stock options of Ironwood Pharmaceuticals . Chien:Ironwood Pharmaceuticals: Employment, Equity Ownership. Currie:Ironwood Pharmaceuticals: Employment, Equity Ownership. Graul:Ironwood Pharmaceuticals: Employment, Other: own stock options of Ironwood Pharmaceuticals .

Soluble cluster of differentiation 74 regulates lung inflammation through the nuclear factor-κB signaling pathway

The soluble form of the migration inhibitory factor receptor cluster of differentiation 74 (sCD74) has previously been shown to be elevated during the development of acute lung injury (ALI) in mice. However, the biological role of increased sCD74 in ALI remans poorly understood. Synthesized recombinant sCD74 protein was administered to mice intratracheally. Pro-inflammatory genes in lung tissue and the inflammatory factors in bronchoalveolar lavage fluid (BALF) were analyzed using RT-PCR and ELISA, respectively. Additionally, RAW264.7, A549, and human umbilical vein endothelial cells (HUVEC) were treated with sCD74, and the resulting pro-inflammatory factor protein and gene expression levels were analyzed in the supernatants and cell lysates. Meanwhile, the level of nuclear factor (NF)-κB components in cell lysates after stimulating macrophages with sCD74 was also assessed. After administration of 0.5 mg/kg body weight sCD74 to mice, the expression of Tnfa, Mip2, and Il6 increased in lung tissues after 2 h by 2.1-, 3.4-, and 2.8-fold, respectively. Moreover, the BALF concentrations of TNF-α and MIP-2 reached maximal levels of 560 and 107 pg/mL at 8 h compared to those in the saline group, respectively. Similarly, TNFA, MIP2, and IL6 expression increased by 4.0-, 11.8-, and 2.6-fold, respectively, 2 h after stimulating macrophages with 1000 ng/mL sCD74. The levels of phospho-IκB and phospho-p65 were also significantly increased in the cytoplasm and nucleus of macrophages following sCD74 stimulation. Taken together, these results suggest that sCD74 is a critical cellular factor involved in the lung acute inflammatory response through nuclear factor-κB signaling.

The non-antibiotic macrolide EM900 attenuates HDM and poly(I:C)-induced airway inflammation with inhibition of macrophages in a mouse model

ObjectiveMacrolides have been reported to reduce the exacerbation of severe asthma. The aim of this study was to clarify the effects and mechanisms of EM900, a non-antibiotic macrolide, on allergic airway inflammation.MethodsMice were sensitized and challenged by house dust mite (HDM), then exposed to polyinosinic-polycytidylic acid (poly(I:C)) as a model of asthma complicated with viral infection. Mice were administered with EM900. Airway inflammation was assessed from inflammatory cells in bronchoalveolar lavage fluid (BALF) and cytokines in lung tissues. Lung interstitial macrophages were counted by flow cytometry. Cytokine production, phosphorylation of NF-κB, and p38 in macrophages were examined by ELISA and western blotting.ResultsCounts of cells in BALF and concentrations of IL-13, IL-5, RANTES, IL-17A, and MIP-2 were significantly decreased by EM900 compared to those without EM900. Percentages of lung interstitial macrophages were significantly decreased with EM900. Concentrations of IL-6, RANTES, and MIP-2 induced by HDM and poly(I:C) were significantly suppressed by EM900 through the suppression of NF-κB and p38 phosphorylation in macrophages.ConclusionsHDM and poly(I:C)-induced airway inflammation is attenuated by EM900 with the inhibition of lung interstitial macrophages. Clinical use of EM900 is expected, because EM900 has inhibitory effects against airway inflammation without inducing bacterial drug resistance.

Liposomal Amphotericin B Fosters the Corticosteroids' Anti-inflammatory Effect on Murine Allergic Bronchopulmonary Aspergillosis Model Airways.

Fungus is an antigen for bronchial asthma causing allergic bronchopulmonary mycosis (ABPM). As a therapy other than corticosteroids, itraconazole (ITCZ) is known to suppress the allergic inflammation induced by Aspergillus fumigatus (Af). However, the efficacy of liposomal amphotericin B (LAMB) with/without corticosteroid on ABPM is unknown. Mice sensitized to Dermatophagoides farinae (Df) allergen were intranasally infected with Af (DfAf group). After the infection, corticosteroid (dexamethasone (Dex)) was administered for 5days (DfAf/Dex group). The effects of ITCZ or LAMB with/without Dex were also evaluated. Pathologically, Dex and LAMB combination treatment decreased the allergic inflammation evidently. The bronchoalveolar lavage fluid (BALF) concentrations of IL-5, IL-13, and MIP-2 were significantly elevated in DfAf mice compared with control mice (p < 0.05, each). In DfAf mice, ITCZ and LAMB significantly decreased the elevation of MIP-2 (p < 0.05 vs the DfAf group). The addition of both Dex and LAMB suppressed the MIP-2 elevation in DfAf mice (p < 0.05 vs the Df/Af/Dex/LAMB group), but the addition of Dex and ITCZ did not (DfAf/Dex/ITCZ group). None of Dex, ITCZ, or LAMB decreased pulmonary IL-13 concentration. It was suggested that combination of antifungal drugs and corticosteroid enhanced the suppressing effect of airway inflammations. This finding will give a hope for the treatment of severe fungus-related asthma.

Optimizing steroid administration in murine models of Citrobacter rodentium-induced acute phase colitis

Abstract This study used Citrobacter rodentium-infected C57BL/6 mice as a model of acute colitis to assess treatment with steroids prednisolone (pred) and dexamethasone (dex). Both germ-free (GF) and normal flora (NF) adult mice were used to determine an optimal steroid dosing strategy to mitigate intestinal inflammation caused by C. rodentium attachment/effacement lesions. Male and female mice (6-weeks of age) were orally inoculated with C. rodentium (1.0 × 108 CFU/100 μL) for two consecutive days. Five days post-infection, mice were gavaged with pred (1 mg/kg/d or 3 mg/kg/d), dex (0.6 mg/kg/d or 1.5 mg/kg/d) or controls for five days (GF n = 3 and NF n = 4 for each treatment). GF mice had longer colons (base of cecum to anus) than NF mice (7.1 ± 0.1 cm vs. 6.2 ± 0.2 cm; P &amp;lt; 0.001) and lighter total liver weight than NF mice (1.0 ± 0 g vs. 1.1 ± 0.2 g; P = 0.03). Colonic cytokines were quantified by ELISA. Steroid treatment decreased concentrations of colonic KC (CXCL1; P &amp;lt; 0.001) in NF mice; and high-dose dex decreased KC more effectively than low-dose dex (3207.5 ± 583.5 pg/g vs. 9217.6 ± 1375.4 pg/g; P = 0.019). Although C. rodentium increased colonic concentrations of KC (P &amp;lt; 0.001), MIP-2 (CXCL2; P = 0.19), TNF-α (P = 0.031), IL-22 (P = 0.018) and myeloperoxidase (P &amp;lt; 0.001) in NF mice, no differences were attributable to steroids or sex in GF and NF mice. Colonic MIP-2 (P &amp;lt; 0.001), IL-10 (P = 0.032), TNF-α (P &amp;lt; 0.001), IL-17 (P &amp;lt; 0.001) and IL-22 (P = 0.005) were higher in NF than GF mice, likely due to underdeveloped gut-associated lymphoid tissue in GF mice. In summary, high-dose dex (1.5 mg/kg/d) but not high-dose pred (3 mg/kg/d) mitigated production of the potent neutrophil chemoattractant KC in a C. rodentium induced model of acute-phase colitis.

Trovafloxacin-Induced Liver Injury: Lack in Regulation of Inflammation by Inhibition of Nucleotide Release and Neutrophil Movement.

The fluoroquinolone trovafloxacin (TVX) is associated with a high risk of drug-induced liver injury (DILI). Although part of the liver damage by TVX+TNF relies on neutrophils, we have recently demonstrated that liver recruitment of monocytes and neutrophils is delayed by TVX. Here we show that the delayed leukocyte recruitment is caused by a combination of effects which are linked to the capacity of TVX to block the hemichannel pannexin 1. TVX inhibited find-me signal release in apoptotic HepG2 hepatocytes, decelerated freshly isolated human neutrophils toward IL-8 and f-MLF, and decreased the liver expression of ICAM-1. In blood of TVX+TNF-treated mice, we observed an accumulation of activated neutrophils despite an increased MIP-2 release by the liver. Depletion of monocytes and neutrophils caused increased serum concentrations of TNF, IL-6, and MIP-2 in TVX-treated mice as well as in mice treated with the fluoroquinolone levofloxacin, known to have a lower DILI-inducing profile. This supports the idea that early leukocyte recruitment regulates inflammation. In conclusion, disrupted regulation by leukocytes appears to constitute a fundamental step in the onset of TVX-induced liver injury, acting in concert with the capability of TVX to induce hepatocyte cell death. Interference of leukocyte-mediated regulation of inflammation represents a novel mechanism to explain the onset of DILI.

Glycyrrhizin inactivates toll-like receptor (TLR) signaling pathway to reduce lipopolysaccharide-induced acute lung injury by inhibiting TLR2.

This study aimed to explore glycyrrhizin on acute lung injury (ALI) and how glycyrrhizin (GL) attenuated lipopolysaccharide (LPS)-induced ALI. Bioinformatics analysis was performed to screen the expressed genes in LPS-induced ALI mice. The enrichment of functions and signaling pathways of deregulated genes were conducted. Combined with DIGSEE and STICH, the target gene for further investigation was chosen. To verify target gene in mice, we performed experiment in vivo. Forty mice were randomized into NC, LPS, LPS + S, and LPS + GL group. Mice in the LPS + GL group received glycyrrhizin l mg and mice in LPS + S received saline. Then, HE and Masson staining detected pathological changes of lung tissues; enzyme-linked immunosorbent assay analyzed bronchoalveolar lavage fluid concentrations of MIP-2, mice growth-related oncogene homologue (KC), IL-4, IL-6, GM-CSF, IFN-γ, and IgM; western blot analysis determined the expression of toll-like receptor (TLR) signaling and NF-κB pathway-related proteins. Tlr2 which was not only upregulated but also closely related to glycyrrhizin. TLR2 was upregulated in following LPS induced in cells and TLR2 overexpression-activated TLR signaling pathway to promote ALI. After glycyrrhizin treatment, the expression of TLR2 was reduced. Furthermore, it was found out that the number of inflammatory cells, collagen deposition, MIP-2, KC, IL-4, IL-6, GM-CSF, and IFN-γ expression increased in ALI mice and glycyrrhizin mitigated it. Similarly, the expression of TLR signaling pathway and NF-κB pathway-related protein also increased. Glycyrrhizin functioned as a suppressor in TLR signaling pathway to reduce LPS-induced ALI by inhibiting TLR2.

Middle age exacerbates acute respiratory distress syndrome in a double hit murine model

RationaleIn a recent systematic review, aging has been identified as the only factor independently associated with mortality during human acute respiratory distress syndrome (ARDS). We explored this age-dependent severity in a clinically relevant double hit murine ARDS model. MethodsYoung adult (Y, 10–12weeks) and middle-old (O, 12–13months) male C57BL6 mice underwent an aspiration of Escherichia coli lipopolysaccharide (LPS) or control saline vehicle. Twenty hours later, four groups of mice were sacrificed [Y(control), O(control), Y(LPS) and O(LPS)]. Four other groups of mice underwent 3h of low tidal volume (8mL/kg) mechanical ventilation (MV) [Y(MV), O(MV), Y(LPS+MV) and O(LPS+MV)]. Lung mechanics were assessed hourly during MV. Right ventricular pressure and cardiac output were measured at the end of the MV. After sacrifice, lung inflammation, edema and injury were explored with bronchoalveolar lavage (BAL) and histology. ResultsAfter saline aspiration, middle-old mice had a higher respiratory system compliance than young adult mice. LPS aspiration dramatically altered the baseline compliance in middle-old (O(LPS)), but not in young adult (Y(LPS)) mice. Middle-old mice had a more pronounced alteration in lungs mechanics during MV as compared to young adult mice. Lung inflammation (as assessed by the total cell count, IL-6, TNFα and MIP-2 concentrations in BAL fluid), systemic inflammation (as assessed by plasma IL-6 concentration) and alveolocapillary leak (as assessed by the total protein concentration of BAL fluid) were higher in O(LPS) and O(LPS+MV) mice as compared to Y(LPS) and Y(LPS+MV) mice, respectively. The combination of LPS+MV induced a higher lung injury as compared to LPS alone in middle-old mice but not in young adult mice. Hemodynamics (systemic blood pressure, cardiac output and pulmonary vascular resistances) were similar between Y(MV) and O(MV) on the one hand and between Y(LPS+MV) and O(LPS+MV) on the other hand. ConclusionMiddle-old mice were more susceptible to both LPS alone and the combination of LPS and low tidal volume MV as compared to their young adult counterparts. The synergism between LPS and MV was amplified in middle-old mice.

Antimicrobial and immunomodulatory effects of tedizolid against methicillin-resistant Staphylococcus aureus in a murine model of hematogenous pulmonary infection

Tedizolid (TZD) is a second-generation oxazolidinone and demonstrates potent in-vitro activity against multidrug-resistant Gram-positive bacteria. Phase III studies in patients with acute bacterial skin and skin structure infections (ABSSSI) have demonstrated the non-inferiority of TZD to linezolid (LZD). However, there are only a few studies that show the effect of TZD in pulmonary infections. In this study, we investigated the effect of TZD in a murine model of hematogenous pulmonary infection caused by methicillin-resistant Staphylococcus aureus (MRSA). The mice were treated either twice daily with saline (control), 25mg/kg of vancomycin (low-VAN), 110mg/kg of vancomycin (high-VAN), 120mg/kg of LZD or once daily with 20mg/kg of TZD. As compared to the control, the low- and high-VAN treatment groups, LZD and TZD significantly improved the survival rate, reduced the bacterial count in the lungs. Furthermore, TZD decreased the area of central bacterial colony zone (CBCZ) at 36h post-inoculation, compared with the control. In addition, we investigated the immunomodulatory effect of TZD by evaluating the plasma concentrations of the inflammatory cytokines. Although there were no significant differences in the bacterial count in the lungs amongst the drugs at 26h post-inoculation, TZD and LZD significantly improved the plasma concentrations of TNF-alpha, IL-6 and MIP-2, in comparison with the control. In this study, both TZD and LZD demonstrated antimicrobial and immunomodulatory efficacy in a murine model of hematogenous pulmonary infection caused by MRSA.

Effect of crossing C57BL/6 and FVB mouse strains on basal cytokine expression.

C57BL/6 is the most often used laboratory mouse strain. However, sometimes it is beneficial to cross the transgenic mice on the C57BL/6 background to the other strain, such as FVB. Although this is a common strategy, the influence of crossing these different strains on homeostatic expression of cytokines is not known. Here we have investigated the differences in the expression of selected cytokines between C57BL/6J and C57BL/6JxFVB mice in serum and skeletal muscle. We have found that only few cytokines were altered by crossing of the strains. Concentrations of IL5, IL7, LIF, MIP-2, and IP-10 were higher in serum of C57BL/6J mice than in C57BL/6JxFVB mice, whereas concentration of G-CSF was lower in C57BL/6J. In the skeletal muscle only the concentration of VEGF was higher in C57BL/6J mice than in C57BL/6JxFVB mice. Concluding, the differences in cytokine expression upon crossing C57BL/6 and FVB strain in basal conditions are not profound.

The role of CXC chemokine receptor 2 in Staphylococcus aureus keratitis

Staphylococcus aureus is a leading cause of corneal infection. CXC receptor 2 binding chemokines have been implicated in the pathogenesis of Pseudomonas aeruginosa keratitis. The role of this receptor in immune responses during Staphylococcus keratitis remains to be fully understood. Corneas of CXC receptor 2 knockout and wild-type mice (Cmkar −/− & Cmkar +/+) were scratched and 1 × 108 cfu/ml of strain Staph 38 applied. Twenty-four hours post-infection, mice were sacrificed and eyes harvested for enumeration of bacteria and measurement of myeloperoxidase levels. Production of inflammatory mediators, cellular adhesion molecules and chemokines in response to infection were investigated by ELISA, and PCR. 24 h after challenge with S. aureus, Cmkar −/− mice had developed a more severe response with a 50-fold higher bacterial load than WT mice. PMNs failed to penetrate the corneas of Cmkar −/− mice. However, concentrations of KC, MIP-2, IL-1β and IL-6 were significantly elevated (6–13 fold) in Cmkar−/− mice. The concentration of LTB4 was decreased (2 fold). Cmkar−/− mice failed to upregulate mRNA for VCAM-1 or PECAM-1 in response to infection, but had constitutively higher levels of ICAM-1. A lack of CXC receptor 2 lead to an inability to control bacterial numbers as a result of failure of PMNs to penetrate the cornea to the site of infection, even when chemokines were more highly produced. These results imply that CXCR2-mediated signaling through upregulation of adhesion molecules is essential to margination of PMNs in this infection model.

Genetic and pharmacologic inhibition of Tpl2 kinase is protective in a mouse model of ventilator-induced lung injury.

BackgroundMechanical stress induced by injurious ventilation leads to pro-inflammatory cytokine production and lung injury. The extracellular-signal-regulated-kinase, ERK1/2, participates in the signaling pathways activated upon mechanical stress in the lungs to promote the inflammatory response. Tumor progression locus 2 (Tpl2) is a MAP3kinase that activates ERK1/2 upon cytokine or TLR signaling, to induce pro-inflammatory cytokine production. The role of Tpl2 in lung inflammation, and specifically in the one caused by mechanical stress has not been investigated. The aim of the study was to examine if genetic or pharmacologic inhibition of Tpl2 could ameliorate ventilator-induced lung injury.MethodsAdult male wild-type and Tpl2-deficient mice were ventilated with normal or high tidal volume for 4 h. Additional wild-type mice were treated with a Tpl2 inhibitor either before or 30 min after initiation of high tidal ventilation. Non-ventilated mice of both genotypes served as controls. The development of lung injury was evaluated by measuring lung mechanics, arterial blood gases, concentrations of proteins, IL-6, and MIP-2 in bronchoalveolar lavage fluid (BALF) and by lung histology. Data were compared by Kruskal-Wallis non-parametric test and significance was defined as p < 0.05.ResultsMechanical ventilation with normal tidal volume induced a mild increase of IL-6 in BALF in both strains. High tidal volume ventilation induced lung injury in wild-type mice, characterized by decreased lung compliance, increased concentrations of proteins, IL-6 and MIP-2 in BALF, and inflammatory cell infiltration on histology. All indices of lung injury were ameliorated in Tpl2-deficient mice. Wild-type mice treated with the Tpl2 inhibitor, either prior of after the initiation of high tidal volume ventilation were protected from the development of lung injury, as indicated by preserved lung compliance and lower BALF concentrations of proteins and IL-6, than similarly ventilated, untreated wild-type mice.ConclusionsGenetic and pharmacologic inhibition of Tpl2 is protective in a mouse model of ventilator-induced lung injury, ameliorating both high-permeability pulmonary edema and lung inflammation.

Role of Nicotinic and Muscarinic Cholinoreceptors in the Realization of the Cholinergic Anti-Infl ammatory Pathway during the Early Phase of Sepsis

Stimulation of nicotinic and muscarinic cholinoreceptors (nAChR, mAChR) in outbred albino mice with nicotine and aceclidine, respectively, in single equilethal doses 0.5 DL(50)6 h before sepsis induction significantly reduced animal mortality due to a decrease in blood concentrations of proinflammatory cytokines IL-1β, IL-6, and MIP-2. Stimulation of mAChR (injection of aceclidine) stimulated the neutrophilic phagocytic and metabolic activity. Realization of the cholinergic anti-inflammatory pathway (stimulation of the peripheral nicotinic cholinoreceptors (α7nAChR) and central muscarinic cholinoreceptors (mAChR) was modulated by stimulation of the muscarinic cholinoreceptors of the phagocytic monocytic system cells.

Efficacy of ceftolozane in a murine model of Pseudomonas aeruginosa acute pneumonia: in vivo antimicrobial activity and impact on host inflammatory response

To assess the activity of ceftolozane, a novel oxyimino-cephalosporin, in comparison with ceftazidime and piperacillin/tazobactam against a multidrug-resistant Pseudomonas aeruginosa strain using a murine model of pneumonia. Quantitative bacteriology, survival, histological examination, myeloperoxidase activity, proinflammatory cytokine levels in lungs and endothelial permeability were evaluated to determine the effects of ceftolozane and comparators on P. aeruginosa-induced pneumonia. After 48 h of treatment, ceftolozane reduced the bacterial load by 3-4 log(10) cfu/g of lung. Systemic dissemination of the pulmonary infection and development of lung damage were inhibited in all β-lactam-treated animals. P. aeruginosa-induced pneumonia led to elevated concentrations of tumour necrosis factor-α, interleukin (IL)-1β and macrophage inflammatory protein (MIP)-2 in the lungs. While the levels of proinflammatory cytokines decreased following ceftazidime and piperacillin/tazobactam therapy, ceftolozane exhibited increased concentrations of IL-1β and MIP-2 after 24 h of infection, resulted in significantly increased levels of recruited neutrophils within the infected lung without increasing lung endothelial permeability. These data strongly support ceftolozane as an effective option for the treatment of severe P. aeruginosa respiratory infections by improving the early pulmonary inflammatory response without impairing 48 h post-infection homeostasis.

Cantharidin-induced inflammation in mouse ear model for translational research of novel anti-inflammatories

The murine model of cantharidin-induced ear inflammation was profiled in detail forits alignment with the human model and to explore the mechanism of anti-inflammatory activity of the macrolide antibiotics, clarithromycin and azithromycin. Ear swelling in CD1 mice persisted for 7 days, with peak intensity at 16 h after inflammation induction. As in humans, cantharidin (12.5 μg/ear) generated macrophage-inflammatory protein (MIP)-2, monocyte chemoattractant protein (MCP)-1, keratinocyte-derived chemokine (KC), interleukin (IL)-6, IL-1β, and myeloperoxidase (MPO) production, as well as neutrophil accumulation in mouse ear tissue. The tested macrolides, clarithromycin and azithromycin, administered orally (2 × 150 mg/kg) 0.5 h before and 5 h after cantharidin challenge, reduced MIP-2, MCP-1, KC, and MPO concentrations and thereby decreased ear swelling. Our results suggest thatcantharidin-induced acute inflammation represents an excellent model for translational research of novel anti-inflammatories.

Inhibition of Pyk2 blocks lung inflammation and injury in a mouse model of acute lung injury

BackgroundProline-rich tyrosine kinase 2 (Pyk2) is essential in neutrophil degranulation and chemotaxis in vitro. However, its effect on the process of lung inflammation and edema formation during LPS induced acute lung injury (ALI) remains unknown. The goal of the present study was to determine the effect of inhibiting Pyk2 on LPS-induced acute lung inflammation and injury in vivo.MethodsC57BL6 mice were given either 10 mg/kg LPS or saline intratracheally. Inhibition of Pyk2 was effected by intraperitoneal administration TAT-Pyk2-CT 1 h before challenge. Bronchoalveolar lavage analysis of cell counts, lung histology and protein concentration in BAL were analyzed at 18 h after LPS treatment. KC and MIP-2 concentrations in BAL were measured by a mouse cytokine multiplex kit. The static lung compliance was determined by pressure-volume curve using a computer-controlled small animal ventilator. The extravasated Evans blue concentration in lung homogenate was determined spectrophotometrically.ResultsIntratracheal instillation of LPS induced significant neutrophil infiltration into the lung interstitium and alveolar space, which was attenuated by pre-treatment with TAT-Pyk2-CT. TAT-Pyk2-CT pretreatment also attenuated 1) myeloperoxidase content in lung tissues, 2) vascular leakage as measured by Evans blue dye extravasation in the lungs and the increase in protein concentration in bronchoalveolar lavage, and 3) the decrease in lung compliance. In each paradigm, treatment with control protein TAT-GFP had no blocking effect. By contrast, production of neutrophil chemokines MIP-2 and keratinocyte-derived chemokine in the bronchoalveolar lavage was not reduced by TAT-Pyk2-CT. Western blot analysis confirmed that tyrosine phosphorylation of Pyk2 in LPS-challenged lungs was reduced to control levels by TAT-Pyk2-CT pretreatment.ConclusionsThese results suggest that Pyk2 plays an important role in the development of acute lung injury in mice and that pharmacological inhibition of Pyk2 might provide a potential therapeutic strategy in the pretreatment for patients at imminent risk of developing acute lung injury.

Impact of aging on pulmonary responses to acute ozone exposure in mice: role of TNFR1

Context: Chamber studies in adult humans indicate reduced responses to acute ozone with increasing age. Age-related changes in TNFα have been observed. TNFα induced inflammation is predominantly mediated through TNFR1.Objective: To examine the impact of aging on inflammatory responses to acute ozone exposure in mice and determine the role of TNFR1 in age-related differences.Materials and methods: Wildtype and TNFR1 deficient (TNFR1−/–) mice aged 7 or 39 weeks were exposed to ozone (2 ppm for 3 h). Four hours after exposure, bronchoalveolar lavage (BAL) was performed and BAL cells, cytokines, chemokines, and protein were examined.Results: Ozone-induced increases in BAL neutrophils and in neutrophil chemotactic factors were lower in 39- versus 7-week-old wildtype, but not (TNFR1−/–) mice. There was no effect of TNFR1 genotype in 7-week-old mice, but in 39-week-old mice, BAL neutrophils and BAL concentrations of MCP-1, KC, MIP-2, IL-6 and IP-10 were significantly greater following ozone exposure in TNFR1−/– versus wildtype mice. BAL concentrations of the soluble form of the TNFR1 receptor (sTNFR1) were substantially increased in 39-week-old versus 7-week-old mice, regardless of exposure.Discussion and conclusion: The data suggest that increased levels of sTNFR1 in the lungs of the 39-week-old mice may neutralize TNFα and protect these older mice against ozone-induced inflammation.

Effect of peroxisome proliferator-activated receptor α agonist on mechanical ventilation-induced lung injury in rats

Objective To investigate the effect of peroxisome proliferator-activated receptor α agonist Wy14643 on mechanical ventilation-induced lung injury in rats.Methods Thirty-two healthy male SD weighing 250-300 g were randomly divided into 4 groups(n = 8 each): group Ⅰ control(group C);group Ⅱ mechanical ventilation(group V)and group Ⅲ,Ⅳ pretreated with different doses of Wy14643(group W1 ,W2).The animals were anesthetized with intraperitoneal 3% pentobarbital 30 mg/kg and tracheostomized.The femoral artery and external jugular vein were cannulated for blood sampling and drug administration.Group C received no mechanical ventilation.In group V,W1 and W2 the animals were mechanically ventilated for 2 h(VT 40 ml/kg,RR40 bpm,I:E=1:2,FiO2 21%).In group W1 and W2 Wy14643 1 and 3 mg/kg were administered iv at 1 h before mechanical ventilation.Arterial blood samples were collected at 1 and 2 h of mechanical ventilation for determination of PaO2/FiO2.Serum SOD activity and MDA concentration were measured at the end of 2 h mechanical ventilation.The animals were then killed and the lungs removed for microscopic examination,lung lavage and W/D lung weight ratio.The MIP-2 and TNF-α concentrations in BALF were measured.Results Two hour mechanical ventilation significantly decreased serum SOD activity and increased serum MDA concentration,W/D lung weight ratio and TNF-α and MIP-2 concentrations in BALF as compared with group C.Wy14643 pretreatment significantly attenuated these mechanical ventilation-induced changes in group W1 and W2 in a dose-dependent manner.Conclusion Wy14643 can attenuate mechanical ventilation induced lung injury in rats and it is related to the dose. Key words: PPAR alpha; Respiratory distress syndrome,adult; Respiration,artificial

Osteoblasts are a major source of inflammatory cytokines in the tumor microenvironment of bone metastatic breast cancer

Metastatic breast cancer cells co-opt the cells of the bone to increase their production of inflammatory cytokines. Here, we sought to identify key cytokines expressed by osteoblasts in vitro and in vivo in the presence of MDA-MB-231 metastatic breast cancer cells, including a bone-seeking variant. We hypothesized that osteoblast-derived cytokines increase in the presence of metastatic breast cancer cell conditioned medium (CM), act as chemoattractants for cancer cells, and enhance osteoclast formation. We detected increases in the concentrations of osteoblast-derived IL-6, MCP-1, VEGF, MIP-2, and KC in vitro in culture supernatants from MC3T3-E1 cells in the presence of metastatic breast cancer cell CM and from cancer-bearing femurs ex vivo. A comparison of cancer cell- and osteoblast-derived cytokines revealed that while breast cancer cells expressed the same or equivalent cytokines as the osteoblasts, the breast cancer cells only produced picogram quantities of MCP-1; osteoblasts expressed nanogram amounts. Bone-derived MCP-1 increased in the proximal metaphysis, an area where breast cancer cells preferentially trafficked following intracardiac inoculation in athymic mice. An MDA-MB-231 bone-seeking variant was not different from parental lines. Osteoblast CM was a potent chemoattractant for metastatic breast cancer cells. Furthermore, culture supernatants of osteoblasts treated with breast cancer cell CM enhanced osteoclast formation. These findings suggest that bone metastatic breast cancer cells utilize osteoblast-derived cytokines to facilitate breast cancer cell colonization and survival upon arrival in the bone microenvironment.