Animal Models Of Lung Inflammation Research Articles

Abstract 2056: PI3Kgamma inhibition reduces inflammation and promotes survival in cancer and SARS-CoV-2

Abstract Solid tumors are characterized by extensive immune suppressive inflammation, vascular leak, fibrosis and organ damage. Similarly, SARS-CoV-2 infections induce aberrant pulmonary and systemic inflammation, vascular leak, coagulation, fibrosis and fatal organ damage. We previously demonstrated that macrophages in solid tumors strongly expressed phosphatidylinositol 3-kinase gamma (PI3Kγ), a signaling protein that coordinately controls granulocyte and monocyte trafficking to tumors as well as wound-healing-type macrophage transcription in cancer and fibrosis. We also observed that macrophages in COVID-19 lungs strongly expressed PI3Kγ. To identify therapeutic strategies to suppress COVID-19-associated inflammation, we characterized lung tissue of COVID-19 patients using multiplex immunohistochemistry and tissue transcriptomics. Lungs of deceased patients exhibited substantial infiltration by neutrophils and wound-healing macrophages, fibrosis and alveolar type II cell depletion. In animal models of lung inflammation, bacterial infections, viral infection and SARS-CoV-2 infection, PI3Kγ deletion or inhibition with the cancer therapeutic IPI-549 (eganelisib) suppressed pulmonary and systemic inflammation, reduced lung damage, and promoted survival. These studies demonstrate the essential role of PI3Kγ in inflammatory diseases as well as cancer and support the use of PI3Kγ inhibitors such as eganelisib to suppress inflammation and promote survival in pulmonary infections like SARS-CoV-2 and cancer. Citation Format: Judith A. Varner, Anghesom Ghebremedhin. PI3Kgamma inhibition reduces inflammation and promotes survival in cancer and SARS-CoV-2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2056.

Is the anti-filarial drug diethylcarbamazine useful to treat COVID-19?

SARS-CoV-2 virus has resulted in a devastating pandemic of COVID-19. Exploring compounds that could offer a breakthrough in treatment is the need of the hour. Re-positioning cheap, freely available and safe drugs is a priority. The paper proposes evidence for the potential use of diethylcarbamazine (DEC) in the treatment of COVID-19. DEC has inhibitory effects on arachidonic acid metabolism to prostaglandins, little known anti-viral effects on animal retroviruses and demonstrated anti-inflammatory actions in animal models of lung inflammation indicating the need to explore this hypothesis further. We believe this is the first time DEC is being proposed to treat COVID-19.

Therapeutic Potential of the Rhizomes of Anemarrhena asphodeloides and Timosaponin A-III in an Animal Model of Lipopolysaccharide-Induced Lung Inflammation

Investigations into the development of new therapeutic agents for lung inflammatory disorders have led to the discovery of plant-based alternatives. The rhizomes of Anemarrhena asphodeloides have a long history of use against lung inflammatory disorders in traditional herbal medicine. However, the therapeutic potential of this plant material in animal models of lung inflammation has yet to be evaluated. In the present study, we prepared the alcoholic extract and derived the saponin-enriched fraction from the rhizomes of A. asphodeloides and isolated timosaponin A-III, a major constituent. Lung inflammation was induced by intranasal administration of lipopolysaccharide (LPS) to mice, representing an animal model of acute lung injury (ALI). The alcoholic extract (50–200 mg/kg) inhibited the development of ALI. Especially, the oral administration of the saponin-enriched fraction (10–50 mg/kg) potently inhibited the lung inflammatory index. It reduced the total number of inflammatory cells in the bronchoalveolar lavage fluid (BALF). Histological changes in alveolar wall thickness and the number of infiltrated cells of the lung tissue also indicated that the saponin-enriched fraction strongly inhibited lung inflammation. Most importantly, the oral administration of timosaponin A-III at 25–50 mg/kg significantly inhibited the inflammatory markers observed in LPS-induced ALI mice. All these findings, for the first time, provide evidence supporting the effectiveness of A. asphodeloides and its major constituent, timosaponin A-III, in alleviating lung inflammation.

Therapeutic Potential of Medicinal Plants and Their Constituents on Lung Inflammatory Disorders.

Acute bronchitis and chronic obstructive pulmonary diseases (COPD) are essentially lung inflammatory disorders. Various plant extracts and their constituents showed therapeutic effects on several animal models of lung inflammation. These include coumarins, flavonoids, phenolics, iridoids, monoterpenes, diterpenes and triterpenoids. Some of them exerted inhibitory action mainly by inhibiting the mitogen-activated protein kinase pathway and nuclear transcription factor-κB activation. Especially, many flavonoid derivatives distinctly showed effectiveness on lung inflammation. In this review, the experimental data for plant extracts and their constituents showing therapeutic effectiveness on animal models of lung inflammation are summarized.

The E3 Ubiquitin Ligase Cbl-b Limits Nascent Th9 Differentiation

Introduction: Th9 cells are critical mediators of allergy and anti-cancer immunity. The E3 ubiquitin ligase Cbl-b modulates T cell activation via regulation of the T cell receptor (TCR) activation threshold as well as by inducing TGF-β sensitivity, which is a critical differentiation factor for Th9 differentiation. Even though some evidence shows that Cbl-b impairs Th9 differentiation by targeting IL-4 dependent STAT6 activation, a complete suppression of Th9 differentiation in the absence of both STAT6 and Cbl-b is not achieved, implying the involvement of additional mechanisms. In this study, we evaluate the role of Cbl-b in early stages of TGF-β dependent Th9 differentiation.Methods: Th9 cells were generated from WT and cblb-deficient naïve CD4+ T cells. After maximum 3 days in presence of IL-4, TGF-β and anti-IFN-γ antibodies, differentiation was determined by the quantification of cytokines, mainly IL-9, and that of the two required transcription factors for Th9 differentiation, namely IRF4 and PU.1. Microarray assay revealed gene candidates that were further validated by mRNA and protein expression analysis. The functional role of Cbl-b was tested in a Th9-mediated murine lung allergy model, in which mice were challenged by intratracheal injections of house dust mite (HDM) extracts.Results:cblb-deficient naïve T cells more efficiently differentiate into Th9 cells after 3 days in culture, express in parallel PU.1 more intensively compared to WT Th9 cells, while retaining similar expression levels of IRF4, another important Th9 differentiation factor. Increased IL-9 level is not based on cblb -deficient T cell hyperproliferation, as we show an increased IL-9 production per cell by using combination of CFSE with intracellular IL-9 staining. Microarray analysis revealed that RUNX1, a known transcriptional modulator of PU.1, is more rapidly down-regulated in cblb-deficient Th9 cells compared to WT Th9 cells. Accordingly, knocking down RUNX1 by siRNA in naïve CD4+ T cells and subsequently differentiating them into Th9 cells, also induces higher IL-9 expression at the mRNA and protein levels in RUNX1-depleted Th9 cells compared to control scrambled siRNA-nucleofected Th9 cells. In the HDM murine allergy model, cblb-deficient mice have a higher lung inflammation as mirrored by increased eosinophils in the BAL and in the lungs, as well as by increased IgE production in the blood. These are also paralleled by an increased IL-9 expression level in the lungs of the allergic cblb -deficient mice.Conclusions: Cbl-b critically limits Th9 differentiation and may thus be a potential target to modify Th9 cell generation in allergy or cancer. Future studies will validate the molecular link that exists between Cbl-b and the RUNX1-dependent IL-9 expression as well as the in vivo significance of increased Th9 cell differentiation in cblb-deficient animal models of lung inflammation and cancer. DisclosuresNo relevant conflicts of interest to declare.

The Role of Coagulation in Pulmonary Pathology

Understanding mechanisms that underlie lung disorders is crucial to achieving optimum care and improved outcomes in pulmonary medicine. Extensive investigations have revealed that inflammation displays an active role in the pathogenesis of these diseases. The byproduct of these inflammatory reactions has been shown to propagate pulmonary disease in consonance with alteration in haemostatic balance. It is now apparent that the two phenomena constitute an interwoven relationship with protective but damaging effects, when dysregulated. However, the precise role of coagulation abnormalities in pulmonary pathology is still evolving. A large body of evidence suggests that an imbalance in intra-alveolar procoagulant and fibrinolytic activities occurs in a variety of lung conditions. This imbalance may even herald a number of pulmonary diseases. Its sequelae have been observed in lung parenchyma of humans and in animal models of lung inflammation. As the pathogenesis of coagulation-related lung diseases continues to be unraveled, therapeutic measures to mitigate pulmonary disease-specific coagulopathy are emerging. Current efforts are directed at depicting multifaceted molecules capable of selective but simultaneous interference with relevant aspects of the dual coagulation-fibrinolytic pathway.

Inhaled LPS induces blood release of Clara cell specific protein (CC16) in human beings

Animal models of lung inflammation have validated the plasma 16-kd Clara cell protein (CC16) as a peripheral marker of the permeability of the alveolocapillary barrier. We investigated in human beings whether inhaled LPS induced a rise in airways permeability measured by the plasma changes in CC16. The CC16 was measured in plasma from 15 subjects exposed to LPS by inhalation, during which the kinetics and the dose-response relationship of LPS-induced CC16 were evaluated. Because LPS-induced response involves macrophages activation, the protective effect of oral methylprednisolone was also evaluated. An inhalation of 50 microg LPS induced a significant ( P < .001) rise in CC16 after 6 hours (from 7.24 [+/-0.68] microg/L to 10.69 [+/-0.99] microg/L) that normalized at 24 hours (6.65 [+/-0.33] microg/L). The CC16 response was dose-related, with the no-response threshold 0.5 microg LPS. A 6-day treatment with 20 mg/d methylprednisolone inhibited significantly ( P < .001) the CC16 response to 50 microg LPS. Exposure to LPS by inhalation in healthy subjects induces an intravascular leakage of CC16 that can be blocked by corticosteroids. These observations further validate plasma CC16 as a noninvasive test of the alveolocapillary barrier permeability.

Nitric oxide: a regulator of mast cell activation and mast cell-mediated inflammation.

Nitric oxide (NO) plays diverse roles in physiological and pathological processes. During immune and inflammatory responses, for example in asthma, NO is generated at relatively high and sustained levels by the inducible form of nitric oxide synthase (NOS-2). NOS-2 derived NO regulates the function, growth, death and survival of many immune and inflammatory cell types. In the case of mast cells, NO suppresses antigen-induced degranulation, mediator release, and cytokine expression. The action of NO on mast cells is time dependent, requiring several hours, and noncGMP mediated, most probably involving chemical modification of proteins. NO inhibits a number of mast cell-dependent inflammatory processes in vivo, including histamine mediated vasodilatation, vasopermeation and leucocyte-endothelial cell attachment. In human asthma and animal models of lung inflammation the role of NO is harder to define. However, although there are conflicting data, the balance of evidence favours a predominantly protective role for NO. Mimicking or targeting NO dependent pathways may prove to be a valuable therapeutic approach to mast cell mediated diseases.

Pharmacological profile of a novel phosphodiesterase 4 inhibitor, 4-(8-benzo[1,2,5]oxadiazol-5-yl-[1,7]naphthyridin-6-yl)-benzoic acid (NVP-ABE171), a 1,7-naphthyridine derivative, with anti-inflammatory activities.

We investigated the pharmacology of a new class of phosphodiesterase 4 (PDE4) inhibitor, 6,8-disubstituted 1,7-naphthyridines, by using 4-(8-benzo[1,2,5]oxadiazol-5-yl-[1,7]naphthyridin-6-yl)-benzoic acid (NVP-ABE171) as a representative compound and compared its potency with the most advanced PDE4 inhibitor, undergoing clinical trials, Ariflo [cis-4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl-r-1-cyclohexanecarboxylic acid)]. NVP-ABE171 inhibited the activity of phosphodiesterase 4A, 4B, 4C, and 4D with respective IC(50) values of 602, 34, 1230, and 1.5 nM. Ariflo was about 40 times less potent. In human cells, NVP-ABE171 inhibited the eosinophil and neutrophil oxidative burst, the release of cytokines by T cells, and the tumor necrosis factor-alpha release from monocytes, in the nanomolar range. Ariflo presented a similar inhibition profile but was 7 to 50 times less potent. In BALB/c mice challenged with lipopolysaccharide, NVP-ABE171 inhibited the airway neutrophil influx and activation with an ED(50) in the range of 3 mg/kg. Ariflo was inactive up to a dose of 10 mg/kg. In ovalbumin sensitized Brown Norway rats, NVP-ABE171 inhibited the lipopolysaccharide-induced airway neutrophil influx and activation (ED(50) of 0.2 mg/kg) and the ovalbumin-induced airway eosinophil influx and activation (ED(50) of 0.1 mg/kg). Ariflo was about 100 times less potent in both models. In the ovalbumin model, NVP-ABE171 had a duration of action of more than 24 h. NVP-ABE171 is a novel PDE4 inhibitor that shows activity both in vitro on human inflammatory cells and in vivo in animal models of lung inflammation. This compound class may have potential for the treatment of airway inflammatory conditions such as asthma and chronic obstructive pulmonary diseases.

Bioactivation of 6,7-dimethyl-2,4-di-1-pyrrolidinyl-7H-pyrrolo[2,3-d]pyrimidine (U-89843) to reactive intermediates that bind covalently to macromolecules and produce genotoxicity.

U-89843 is a novel pyrrolo[2,3-d]pyrimidine antioxidant with prophylactic activity in animal models of lung inflammation. During preclinical safety evaluation, U-89843 was found to give a positive response in the in vitro unscheduled DNA synthesis (UDS) assay, an assay which measures DNA repair following chemically-induced DNA damage in metabolically competent rat hepatocytes. Incubation of [14C]U-89843 with liver microsomes resulted in covalent binding of radioactive material to macromolecules by a process that was NADPH-dependent. U-89843 has been shown to undergo C-6 methylhydroxylation to give U-97924, in rat both in vivo and in vitro, in a reaction catalyzed by cytochrome P450 2C11. Synthetical U-97924 is chemically reactive and undergoes dimerization in aqueous solution. The dimerization of U-97924 was significantly inhibited by addition of nucleophiles such as methanol, glutathione, and N-acetylcysteine. Characterization of the corresponding methanol, glutathione, and N-acetylcysteine adducts of U-97924 supported the hypothesis of a reaction pathway involving reactive iminium species formed via dehydration of U-97924. The metabolism-dependent irreversible covalent binding of radioactive material to liver microsomal protein and DNA also is dramatically reduced in the presence of reduced glutathione (GSH). A trifluoromethyl analog of U-89843 was prepared in an effort to block the corresponding metabolic hydroxylation pathway. This new compound (U-107634) was found to be negative in the in vitro UDS assay, and its metabolic susceptibility toward hydroxylation at the C-6 methyl group was eliminated. These observations suggest that the positive in vitro UDS results of U-89843 are mediated by the bioactivation of U-89843, leading to reactive electrophilic intermediates derived from the (hydroxymethyl)pyrrole metabolite U-97924.

Molecular Mechanisms in Acute Lung Injury

This chapter explores the experience in the use of lung inflammatory models, which have provided information on the role of various inflammatory mediators and their sequence of engagement. This information has provided insights into potential interventional approaches for the suppression of inflammatory processes in humans. The use of animal models of lung inflammation has allowed for the probing of pathways of inflammation responsible for injury. Although the models do not necessarily extrapolate to lung inflammatory diseases in humans, they have permitted a detailed dissection of events that ultimately lead to damage of interstitial capillary endothelial cells and alveolar epithelial cells. In this chapter, models of inflammatory injury in rat lung are discussed: injury occurring after systemic activation of complement and injury developing after alveolar deposition of either IgG or IgA immune complexes. In each model, there are similarities but also distinctive differences that have provided insights into diverse pathways by which the inflammatory response causes tissue injury. This chapter also demonstrates how the in vitro use of endothelial cell monolayers has provided an approach to understanding the mechanisms by which activated neutrophils injure endothelial cells