Model Of LPS-induced Lung Inflammation Research Articles

Delicate regulation of IL-1β-mediated inflammation by cyclophilin A.

The inflammatory response is tightly regulated, but its regulatory principles are still incompletely understood. Cyclophilin A (CypA) has long been considered as a pro-inflammatory factor. Here, we discover how CypA precisely regulates interleukin-1β (IL-1β)-mediated inflammatory responses. In lipopolysaccharide-treated mice, CypA deficiency initially inhibits and then promotes lung inflammation, which is closely related to IL-1β production. Mechanistically, CypA not only facilitates pro-IL-1β processing by increasing Smurf1-mediated K63-linked ubiquitination in an ATP-dependent manner but also accelerates pro-IL-1β degradation, depending on Smurf1-mediated K48-linked ubiquitination. Moreover, in IL-1β-treated mice, CypA exacerbates lung injury by enhancing cytokine production. It also upregulates the ILK/AKT pathway by inhibiting Cyld-mediated K63-linked ILK deubiquitination, which promotes the epithelial-mesenchymal transition (EMT) to facilitate lung repair. Collectively, CypA promotes inflammation activation by increasing IL-1β production and then promotes inflammation resolution by enhancing redundant pro-IL-1β degradation and IL-1β-induced EMT, indicating the complex and delicate regulation of inflammatory response.

Treatment of acute lung inflammation by pulmonary delivery of anti-TNF-α siRNA with PAMAM dendrimers in a murine model

To improve the efficacy of nucleic acid-based therapeutics, e.g., small interfering RNA (siRNA), transfection agents are needed for efficient delivery into cells. Several classes of dendrimers have been found useful as transfection agents for the delivery of siRNA because their surface can readily be functionalized, and the size of the dendriplexes they form with siRNA is within the range of conventional nanomedicine. In this study, commercially available generation 3 poly(amidoamine) (PAMAM) dendrimer was investigated for pulmonary delivery of siRNA directed against tumor necrosis factor (TNF) α for the treatment of acute lung inflammation. Delivery efficiency was assessed in vitro in the RAW264.7 macrophage cell line activated with lipopolysaccharide (LPS), and efficacy was evaluated in vivo in a murine model of LPS-induced lung inflammation upon pre-treatment with TNF-α siRNA. The PAMAM dendrimer-siRNA complexes (dendriplexes) displayed strong siRNA condensation and high cellular uptake in macrophages compared with non-complexed siRNA. Q-PCR analyses showed that the dendriplexes mediated efficient and specific TNF-α silencing in vitro, as compared to non-complexed siRNA and dendriplexes with negative control siRNA. Also in vivo, the PAMAM dendriplexes induced efficacious TNF-α siRNA inhibition, as compared to non-complexed siRNA, upon pulmonary administration to mice with LPS-induced lung inflammation. Hence, these data suggest that PAMAM dendrimers are promising for the local delivery of TNF-α siRNA in the treatment of lung inflammation via pulmonary administration.

Sea snake cathelicidin (Hc-cath) exerts a protective effect in mouse models of lung inflammation and infection

We investigated the anti-inflammatory and antibacterial activities of Hc-cath, a cathelicidin peptide derived from the venom of the sea snake, Hydrophis cyanocyntus, using in vivo models of inflammation and infection. Hc-cath function was evaluated in in vitro, in vivo in the wax moth, Galleria mellonella, and in mouse models of intraperitoneal and respiratory Pseudomonas aeruginosa infection. Hc-Cath downregulated LPS-induced pro-inflammatory responses in macrophages and significantly improved the survival of P. aeruginosa infected G. mellonella over a 5-day period. We also demonstrated, for the first time, that Hc-cath can modulate inflammation in a mouse model of LPS-induced lung inflammation by significantly reducing the release of the pro-inflammatory cytokine and neutrophil chemoattractant, KC, resulting in reduced cellular infiltration into the lungs. Moreover, Hc-cath treatment significantly reduced the bacterial load and inflammation in mouse models of P. aeruginosa intraperitoneal and respiratory infection. The effect of Hc-cath in our studies highlights the potential to develop this peptide as a candidate for therapeutic development.

PA401, a novel CXCL8-based biologic therapeutic with increased glycosaminoglycan binding, reduces bronchoalveolar lavage neutrophils and systemic inflammatory markers in a murine model of LPS-induced lung inflammation

RationaleNeutrophils play a fundamental role in a number of chronic lung diseases. Among the mediators of their recruitment to the lung, CXCL8 (IL-8) is considered to be one of the major players. CXCL8 exerts its chemotactic activity by binding to its GPCR receptors (CXCR1/R2) located on neutrophils, as well as through interactions with glycosaminoglycans (GAGs) on cell surfaces including those of the microvascular endothelium. Binding to GAG co-receptors is required to generate a solid-phase haptotactic gradient and to present IL-8/CXCL8 in a proper conformation to its receptors on circulating neutrophils. MethodsWe have engineered increased GAG-binding affinity into human CXCL8, thereby obtaining a competitive inhibitor that displaces wild-type IL-8/CXCL8 from GAGs. By additionally knocking-out the GPCR binding domain of the chemokine, we generated a dominant negative protein (dnCXCL8; PA401) with potent anti-inflammatory characteristics proven in vivo in a murine model of LPS-induced lung inflammation (Adage et al., 2015).Here we have further investigated PA401 activity in this pulmonary model by evaluating plasma changes induced by LPS on white blood cells (WBC) and a broad range of inflammatory markers, especially chemokines, by addressing immediate effects of PA401 on these parameters in healthy and LPS exposed mice. ResultsAerosolized LPS induced a significant increase in bronchoalveolar lavage (BAL) neutrophils after 3 and 7h, as well as an increase in total WBC and changes in 21 of the 59 measured plasma markers, mostly belonging to the chemokine family.PA401 treatment in saline exposed mice didn’t induce major changes in any of the measured parameters. When administered to LPS aerosolized mice, PA401 caused a significant normalization of KC/mCXCL1 and other inflammatory markers, as well as of blood WBC count. In addition, BAL neutrophils were significantly reduced, confirming the previously observed lung anti-inflammatory activity of PA401 in this experiment. ConclusionsPA401 is a new promising biologic therapeutic with a novel and unique mechanism of action for interfering with neutrophilic lung inflammation, that also normalizes plasma inflammatory markers.

Proteinase-activated receptor-1, CCL2, and CCL7 regulate acute neutrophilic lung inflammation.

PAR1 plays a central role in mediating the interplay between coagulation and inflammation, but its role in regulating acute neutrophilic inflammation is unknown. We report that antagonism of PAR1 was highly effective at reducing acute neutrophil accumulation in a mouse model of LPS-induced lung inflammation. PAR1 antagonism also reduced alveolar-capillary barrier disruption in these mice. This protection was associated with a reduction in the expression of the chemokines, CCL2 and CCL7, but not the proinflammatory cytokines, TNF and IL-6, or the classic neutrophil chemoattractants, CXCL1 and CXCL2. Antibody neutralization of CCL2 and CCL7 significantly reduced LPS-induced total leukocyte and neutrophil accumulation, recovered from the bronchoalveolar lavage fluid of challenged mice. Immunohistochemical analysis revealed that CCL2 predominantly localized to alveolar macrophages and pulmonary epithelial cells, whereas CCL7 was restricted to the pulmonary epithelium. In keeping with these observations, the intranasal administration of recombinant CCL2 (rCCL2) and rCCL7 led to the accumulation of neutrophils within the lung airspaces of naive mice in the absence of any underlying inflammation. Flow cytometry analysis further demonstrated an increase in Ly6G(hi) neutrophils expressing the chemokine receptors, CCR1 and CCR2, isolated from mouse lungs compared with circulating neutrophils. Conversely, the expression of CXCR2 decreased on neutrophils isolated from the lung compared with circulating neutrophils. Furthermore, this switch in chemokine receptor expression was accentuated after acute LPS-induced lung inflammation. Collectively, these findings reveal a novel role for PAR1 and the chemokines, CCL2 and CCL7, during the early events of acute neutrophilic inflammation.

Characterization of AQX‐1125, a small‐molecule SHIP1 activator

The efficacy of AQX-1125, a small-molecule SH2-containing inositol-5'-phosphatase 1 (SHIP1) activator and clinical development candidate, is investigated in rodent models of inflammation. AQX-1125 was administered orally in a mouse model of passive cutaneous anaphylaxis (PCA) and a number of rodent models of respiratory inflammation including: cigarette smoke, LPS and ovalbumin (OVA)-mediated airway inflammation. SHIP1 dependency of the AQX-1125 mechanism of action was investigated by comparing the efficacy in wild-type and SHIP1-deficient mice subjected to an intrapulmonary LPS challenge. AQX-1125 exerted anti-inflammatory effects in all of the models studied. AQX-1125 decreased the PCA response at all doses tested. Using bronchoalveolar lavage (BAL) cell counts as an end point, oral or aerosolized AQX-1125 dose dependently decreased the LPS-mediated pulmonary neutrophilic infiltration at 3-30 mg kg⁻¹ and 0.15-15 μg kg⁻¹ respectively. AQX-1125 suppressed the OVA-mediated airway inflammation at 0.1-10 mg kg⁻¹. In the smoke-induced airway inflammation model, AQX-1125 was tested at 30 mg kg⁻¹ and significantly reduced the neutrophil infiltration of the BAL fluid. AQX-1125 (10 mg kg⁻¹) decreased LPS-induced pulmonary neutrophilia in wild-type mice but not in SHIP1-deficient mice. The SHIP1 activator, AQX-1125, suppresses leukocyte accumulation and inflammatory mediator release in rodent models of pulmonary inflammation and allergy. As shown in the mouse model of LPS-induced lung inflammation, the efficacy of the compound is dependent on the presence of SHIP1. Pharmacological SHIP1 activation may have clinical potential for the treatment of pulmonary inflammatory diseases.

Adenosine receptor A2b on hematopoietic cells mediates LPS-induced migration of PMNs into the lung interstitium

Uncontrolled transmigration of polymorphonuclear leukocytes (PMNs) into the different compartments of the lungs (intravascular, interstitial, alveolar) is a critical event in the early stage of acute lung injury and acute respiratory distress syndrome. Adenosine receptor A(2b) is highly expressed in the inflamed lungs and has been suggested to mediate cell trafficking. In a murine model of LPS-induced lung inflammation, we investigated the role of A(2b) on migration of PMNs into the different compartments of the lung. In A(2b)(-/-) mice, LPS-induced accumulation of PMNs was significantly higher in the interstitium, but not in the alveolar space. In addition, pulmonary clearance of PMNs was delayed in A(2b)(-/-) mice. Using chimeric mice, we identified A(2b) on hematopoietic cells as crucial for PMN migration. A(2b) did not affect the release of relevant chemokines into the alveolar space. LPS-induced microvascular permeability was under the control of A(2b) on both hematopoietic and nonhematopoietic cells. Activation of A(2b) on endothelial cells also reduced formation of LPS-induced stress fibers, highlighting its role for endothelial integrity. A specific A(2b) agonist (BAY 60-6583) was effective in decreasing PMN migration into the lung interstitium and microvascular permeability. In addition, in vitro transmigration of human PMNs through a layer of human endothelial or epithelial cells was A(2b) dependent. Activation of A(2b) on human PMNs reduced oxidative burst activity. Together, our results demonstrate anti-inflammatory effects of A(2b) on two major characteristics of acute lung injury, with a distinct role of hematopoietic A(2b) for cell trafficking and endothelial A(2b) for microvascular permeability.

Effect of PAR-2 Deficiency in Mice on KC Expression after Intratracheal LPS Administration

Protease activated receptors (PAR) have been shown to play a role in inflammation. PAR-2 is expressed by numerous cells in the lung and has either proinflammatory, anti-inflammatory, or no effect depending on the model. Here, we examined the role of PAR-2 in a model of LPS-induced lung inflammation. We found that PAR-2-deficient mice had significantly less KC expression in bronchial lavage fluid compared with wild-type mice but there was no difference in MIP-2 or TNF-α expression. We also found that isolated alveolar and resident peritoneal macrophages lacking PAR-2 showed a similar deficit in KC after LPS stimulation without differences in MIP-2 or TNF-α. Infiltration of neutrophils and macrophages into the lung following LPS administration was not affected by an absence of PAR-2. Our results support the notion that PAR-2 plays a role in LPS activation of TLR4 signaling in macrophages.

C-Jun NH2-TERMINAL KINASE REGULATES LIPOPOLYSACCHARIDE-INDUCED PULMONARY MONONUCLEAR CELL RECRUITMENT VIA CCL2

Subsequent to the initial recruitment of neutrophils, monocytes are recruited to the lung after an injurious insult. Previously the authors have shown that inhibition of either p38 or c-Jun NH2-terminal kinase (JNK) decreased pulmonary neutrophil recruitment in mice exposed to lipopolysaccharide (LPS). As the signaling pathways regulating the influx of mononuclear cells to the lung are poorly understood, the authors undertook the present study to examine the roles of p38 and JNK. In a model of LPS-induced lung inflammation, systemic inhibition of JNK, but not p38, decreased the recruitment of mononuclear cells to the lung. Levels of CCL2 (monocyte chemoattractant protein 1 [MCP-1]) were decreased in the setting of JNK inhibition, with LPS-induced pulmonary mononuclear cell recruitment in CCL2-deficient mice similar to that found with JNK inhibition. The decrease in LPS-induced CCL2 levels in the lung seen with JNK inhibition, however, was independent of neutrophil recruitment, as systemic depletion of neutrophils had no effect on pulmonary CCL2 levels after LPS exposure. In sum, these results suggest that JNK, but not p38, regulates LPS-induced mononuclear cell recruitment to the lung, that this occurs through a CCL2-dependent pathway, and that LPS-induced pulmonary CCL2 expression is dependent on JNK but independent of pulmonary neutrophil recruitment.

Coumarinic derivatives show anti-inflammatory effects on alveolar macrophages, but their anti-elastase activity is essential to reduce lung inflammation in vivo

We have previously demonstrated the potency of coumarinic derivatives to inhibit human leukocyte elastase. Given the anti-inflammatory activities of some coumarins, we investigated the capacity of our coumarinic derivatives to inhibit inflammation and whether their anti-elastase activity was essential for their anti-inflammatory functions. All compounds studied were coumarinic derivatives displaying differential anti-proteinase activity. Coumarinic derivatives 1, 2, and 3 efficiently inhibited human leukocyte elastase in vitro, whereas the coumarinic derivative 4 did not show inhibitory activity. The anti-inflammatory effect of these compounds and a coumarin control, scopoletin, on interleukin-6 (IL-6), tumor necrosis factor (TNF), and macrophage chemotactic protein-1 (MCP-1) release was studied using lipopolysaccharide (LPS)-stimulated alveolar macrophages. The in vivo effect of compound 2, that inhibits elastase, and compound 4, that does not show proteinase inhibition, was investigated using a mouse model of LPS-induced lung inflammation and elastase-induced acute lung injury. All investigated coumarinic derivatives, regardless of their anti-proteinase activity, significantly inhibited IL-6 and TNF production by LPS-stimulated alveolar macrophages. However, only compounds 2, 3, and 4 significantly reduced MCP-1 release. Compound 2 attenuated LPS-induced leukocyte recruitment in bronchoalveolar lavage, whereas no inhibition was observed with compound 4 devoid of elastase inhibitory capacity. Interestingly, MCP-1 level was reduced in bronchoalveolar lavage of compound 4 treated mice, whereas TNF and IL-6 levels were not modulated by coumarins. Furthermore, compound 2, but not 4, reduced elastase induced lung injury. Our data suggest that although coumarinic derivatives have anti-inflammatory properties, their anti-elastase activity is essential to reduce lung inflammation in vivo.

Anti-Inflammatory Effects of LJP 1586 [Z-3-Fluoro-2-(4-methoxybenzyl)allylamine Hydrochloride], an Amine-Based Inhibitor of Semicarbazide-Sensitive Amine Oxidase Activity

Semicarbazide-sensitive amine oxidase (SSAO, amine oxidase, copper-containing 3, and vascular adhesion protein-1) is a copper-containing enzyme that catalyzes the oxidative deamination of primary amines to an aldehyde, ammonia, and hydrogen peroxide. SSAO is also involved in leukocyte migration to sites of inflammation, and the enzymatic activity of SSAO is essential to this role. Thus, inhibition of SSAO enzyme activity represents a target for the development of small molecule anti-inflammatory compounds. Here, we have characterized the novel SSAO inhibitor, Z-3-fluoro-2-(4-methoxybenzyl)allylamine hydrochloride (LJP 1586), and assessed its anti-inflammatory activity. LJP 1586 is a potent inhibitor of rodent and human SSAO activity, with IC(50) values between 4 and 43 nM. The selectivity of LJP 1586 was confirmed with a broad panel of receptors and enzymes that included the monoamine oxidases A and B. Oral administration of LJP 1586 resulted in complete inhibition of rat lung SSAO, with an ED(50) between 0.1 and 1 mg/kg, and a pharmacodynamic half-life of greater than 24 h. In a mouse model of inflammatory leukocyte trafficking oral dosing with LJP 1586 resulted in significant dose-dependent inhibition of neutrophil accumulation, with an effect comparable to that of anti-leukocyte function-associated antigen-1 antibody. In a rat model of LPS-induced lung inflammation, administration of 10 mg/kg LJP 1586 resulted in a 55% significant reduction in transmigrated cells recovered by bronchoalveolar lavage. The results demonstrate that a selective, orally active small molecule inhibitor of SSAO is an effective anti-inflammatory compound in vivo and provide further support for SSAO as a therapeutic anti-inflammatory target.

TRAF1 regulates recruitment of lymphocytes and, to a lesser extent, neutrophils, myeloid dendritic cells and monocytes to the lung airways following lipopolysaccharide inhalation

Inhaled lipopolysaccharide (LPS) induces an inflammatory response that may contribute to the pathogenesis of asthma and other airway diseases. Here we investigate the role of tumour necrosis factor (TNF) receptor-associated factor 1 (TRAF1) in leucocyte recruitment using a model of LPS-induced lung inflammation in mice. TRAF1(-/-) mice are completely deficient in the recruitment of lymphocytes to the lower respiratory tract after inhalation of LPS. Although TRAF1(-/-) mice display normal early accumulation of neutrophils, dendritic cells and monocytes in the alveolar airspace, they have a significantly reduced recruitment of these cells by 24 hr after inhalation of LPS when compared to wild-type (WT) mice. Despite normal expression of the pro-inflammatory cytokines TNF, interleukin-1 (IL-1) and IL-6 after LPS treatment, TRAF1(-/-) mice displayed decreased expression of intercellular adhesion molecule 1, vascular cell adhesion molecule 1, CCL17 and CCL20 in the lungs, when compared to LPS-treated WT mice. These results suggest that TRAF1 facilitates LPS-induced leucocyte recruitment into the lung airways by augmenting the expression of chemokines and adhesion molecules. Mice lacking TNF receptor 1 (TNFR1) but not TNFR2 show a phenotype similar to the TRAF1(-/-) mice, suggesting that TRAF1 may act downstream of TNFR1. Significantly, we use bone marrow chimeras to demonstrate that expression of TRAF1 by cells resident in the lungs, but not by circulating leucocytes, is necessary for efficient LPS-induced recruitment of leucocytes to the lung airways.

IL-17, produced by lymphocytes and neutrophils, is necessary for lipopolysaccharide-induced airway neutrophilia: IL-15 as a possible trigger.

IL-17 is a cytokine implicated in the regulation of inflammation. We investigated the role of this cytokine in neutrophil recruitment using a model of LPS-induced lung inflammation in mice. In the bronchoalveolar lavage, LPS induced a first influx of neutrophils peaking at day 1, followed by a second wave, peaking at day 2. IL-17 levels were increased during the late phase neutrophilia (day 2), and this was concomitant with an increased number of T cells and macrophages, together with an increase of KC and macrophage-inflammatory protein-2 levels in the lung tissue. Intranasal treatment with a neutralizing murine anti-IL-17 Ab inhibited the late phase neutrophilia. In the bronchoalveolar lavage cells, IL-17 mRNA was detected at days 1, 2, and 3 postchallenge, with a strong expression at day 2. This expression was associated with CD4(+) and CD8(+) cells, but also with neutrophils. When challenged with LPS, despite the absence of T cells, SCID mice also developed a neutrophilic response associated with IL-17 production. In BALB/c mice, IL-15 mRNA, associated mainly with neutrophils, was evidenced 1 day after LPS challenge. In vitro, IL-15 was able to induce IL-17 release from purified spleen CD4(+) cells, but not spleen CD8(+) or airway neutrophils. We have shown that IL-17, produced mainly by CD4(+) cells, but also by neutrophils, plays a role in the mobilization of lung neutrophils following bacterial challenge. In addition, our results suggest that IL-15 could represent a physiological trigger that leads to IL-17 production following bacterial infection.