Small Immunomodulatory Molecules as Potential Therapeutics in Experimental Murine Models of Acute Lung Injury (ALI)/Acute Respiratory Distress Syndrome (ARDS)

Abstract

Background: Acute lung injury (ALI) or its most advanced form, acute respiratory distress syndrome (ARDS) is a severe inflammatory pulmonary process triggered by a variety of insults including sepsis, viral or bacterial pneumonia, and mechanical ventilator-induced trauma. Currently, there are no effective therapies available for ARDS. We have recently reported that a novel small molecule AVR-25 derived from chitin molecule (a long-chain polymer of N-acetylglucosamine) showed anti-inflammatory effects in the lungs. The goal of this study was to determine the efficacy of two chitin-derived compounds, AVR-25 and AVR-48, in multiple mouse models of ALI/ARDS. We further determined the safety and pharmacokinetic (PK) profile of the lead compound AVR-48 in rats.

Methods: ALI in mice was induced by intratracheal instillation of a single dose of lipopolysaccharide (LPS; 100 µg) for 24 h or exposed to hyperoxia (100% oxygen) for 48 h or undergoing cecal ligation and puncture (CLP) procedure and observation for 10 days.

Results: Both chitin derivatives, AVR-25 and AVR-48, showed decreased neutrophil recruitment and reduced inflammation in the lungs of ALI mice. Further, AVR-25 and AVR-48 mediated diminished lung inflammation was associated with reduced expression of lung adhesion molecules with improvement in pulmonary endothelial barrier function, pulmonary edema, and lung injury. Consistent with these results, CLP-induced sepsis mice treated with AVR-48 showed a significant increase in survival of the mice (80%) and improved lung histopathology in the treated CLP group. AVR-48, the lead chitin derivative compound, demonstrated a good safety profile.

Conclusion: Both AVR-25 and AVR-48 demonstrate the potential to be developed as therapeutic agents to treat ALI/ARDS.

Keywords: AVR-25; AVR-48; acute lung injury; lung inflammation; pulmonary edema; sepsis.

Figure 1

AVR-25 or AVR-48 treatment of LPS- and hyperoxia-induced ALI mice ameliorated lung inflammation. (A) Chemical structure of the discussed compounds: Compound AVR-48 is a short chain analog of our previously published compound AVR-25 with IUPAC name as N-((2S,3R,4R,5S,6R)-4,5-dihydroxy-6-(hydroxymethyl)-2-(4-nitrophenoxy)tetrahydro-2H-pyran-3-yl) acetamide. (B,C,H,I) Total inflammatory and neutrophil cell counts in BALF of LPS- and hyperoxia-induced ALI in mice treated with or without AVR-25 or AVR-48 (n = 5–6, * p < 0.05, ** p < 0.01 and *** p < 0.001). (D,E,J,K) ELISA assay for IL-1β and IL-6 in BALF of LPS- and hyperoxia-induced ALI in mice treated with or without AVR-25 or AVR-48 (n = 5–6, * p < 0.05, ** p < 0.01 and *** p < 0.001). (F,G,L,M) Western Blots analysis for IL-1β and IL-6 in the lungs of LPS- and hyperoxia-induced ALI in mice treated with or without AVR-25 or AVR-48. Right panel shows densitometric quantification of the immunoblots (n = 5–6, * p < 0.05, ** p < 0.01 and *** p < 0.001). Data are expressed as mean ± SEM. For statistical analysis, student’s unpaired t-test and one-way analysis of variance (ANOVA) followed by Tukey post hoc analysis were used. LPS: lipopolysaccharide; ALI: acute lung injury; BALF: bronchoalveolar lavage fluid; IL: interleukin; PBS: phosphate buffered saline; RA: room air; HYP: hyperoxia.

Figure 2

AVR-25 or AVR-48 treatment enhances anti-inflammatory cytokine in LPS- and hyperoxia-induced ALI mice. (A,B) ELISA assay for the levels of anti-inflammatory cytokine IL-10 in the BALF (n = 5–6, * p < 0.05 and ** p < 0.01). Data are expressed as mean ± SEM. For statistical analysis, student’s unpaired t-test and one-way analysis of variance (ANOVA) followed by Tukey post hoc analysis were used. LPS: lipopolysaccharide; ALI: acute lung injury; BALF: bronchoalveolar lavage fluid; IL: interleukin; PBS: phosphate buffered saline; RA: room air; HYP: hyperoxia.

Figure 3

AVR-25 or AVR-48 treatment suppresses the production of lung adhesion molecules in in LPS- and hyperoxia-induced ALI mice. (A,D) ELISA assay for the levels of E-selectin in lungs of LPS- and hyperoxia-induced ALI in mice treated with or without AVR-25 or AVR-48 (n = 5–6, * p < 0.05 and ** p < 0.01). (B,C,E,F) Western Blots analysis for ICAM-1, VCAM-1, and E-selectin in the lungs of LPS- and hyperoxia-induced ALI in mice treated with or without AVR-25 or AVR-48. Right panel shows densitometric quantification of the immunoblots (n = 4–6, * p < 0.05, ** p < 0.01 and *** p < 0.001). Data are expressed as mean ± SEM. For statistical analysis, student’s unpaired t-test and one-way analysis of variance (ANOVA) followed by Tukey post hoc analysis were used. LPS: lipopolysaccharide; ALI: acute lung injury; PBS: phosphate buffered saline; RA: room air; HYP: hyperoxia.

Figure 4

AVR-25 or AVR-48 treatment to LPS- and hyperoxia-induced ALI mice showed an improved endothelial barrier function. (A,C) Western blotting showing expression of junctional adherence proteins VE-cadherin, β-catenin, and Src in lungs of LPS- and hyperoxia-induced lung injury in mice treated with or without AVR-25 or AVR-48. (B,D) Densitometric quantification of the immunoblots (n = 4–6, * p < 0.05 ** p < 0.01 and *** p < 0.001). (E–H) Pulmonary edema as measured by total protein concentration in the BALF and Evans Blue dye concentration in the lungs of LPS- and hyperoxia-induced lung injured mice (n = 5–6, * p < 0.05 ** p < 0.01 and *** p < 0.001). Data are expressed as mean ± SEM. For statistical analysis, student’s unpaired t-test and one-way analysis of variance (ANOVA) followed by Tukey post hoc analysis were used. LPS: lipopolysaccharide; ALI: acute lung injury; BAL: bronchoalveolar lavage fluid; PBS: phosphate buffered saline; RA: room air; HYP: hyperoxia.

Figure 5

AVR-25 or AVR-48 treatment attenuates lung cell death in LPS- and hyperoxia-induced ALI mice. (A–D) Western blot analysis for cleaved caspase-3 in lungs of LPS and hyperoxia-induced lung injury in mice treated with or without AVR-25 or AVR-48. Densitometric quantification of the immunoblots is shown right to immunoblots (n = 4–6, * p < 0.05 and ** p < 0.01). (E,F) Representative figure of TUNEL staining (green color) of apoptotic cells and quantification of apoptotic cells in lungs of LPS and hyperoxia-induced lung injury in mice treated with or without AVR-25 or AVR-48 (* p ≤ 0.05). Scale bar = 20 µm. Data are expressed as mean ± SEM. For statistical analysis, student’s unpaired t-test and one-way analysis of variance (ANOVA) followed by Tukey post hoc analysis were used. LPS: lipopolysaccharide; ALI: acute lung injury; PBS: phosphate buffered saline; RA: room air; HYP: hyperoxia.

Figure 6

AVR-25 or AVR-48 treatment attenuated pulmonary injury in LPS- and hyperoxia-induced ALI mice. (A,C) Representative image of hematoxylin and eosin (H/E)-stained lungs of LPS- and hyperoxia-induced lung injury in mice receiving either AVR-25 or AVR-48 treatment or non-treatment (n = 4–5 in each group). Scale bar = 100 µm. (B,D) Lung injury score in LPS- and hyperoxia-induced lung injury in mice receiving AVR-25 or AVR-48 treatment or non-treatment (n = 4–5 in each group, * p < 0.05 ** p < 0.01 and *** p < 0.001). AVR-25 or AVR-48 treated ALI mice showed decreased lung injury score as demonstrated by diminished pulmonary hemorrhage, peri-vascular exudates, thickened alveolar septa, and airspace edema as compared to ALI mice without treatment. Data are expressed as mean ± SEM. For statistical analysis, student’s unpaired t-test and one-way analysis of variance (ANOVA) followed by Tukey post hoc analysis were used. LPS: lipopolysaccharide; ALI: acute lung injury; PBS: phosphate buffered saline; RA: room air; HYP: hyperoxia.

Figure 7

Treatment with AVR-48 showed decreased lung injury in CLP-induced ALI in mice. Representative image of H/E-stained lungs of CLP-induced lung injury in mice receiving AVR-48 treatment. (A) Normal lungs in the sham group. (B) In the CLP group, the alveolar membranes show fragmentation leading to expansion of alveolar sacs associated with severe vascular congestion, thrombosis, and hemorrhage. (C) Signs of lung injury are diminished after treatment with AVR-48 alone or (D) with a combination of imipenem + AVR-48. A CLP + imipenem group was not done here, as we published that finding earlier and observed that AVR-25 demonstrated better efficacy when given in combination with imipenem, than with imipenem alone. Scale bar = 100 µm representative of (A–D). (E) Right panel shows lung injury scores with severe injury in the CLP group followed by significant recovery after treatment with AVR-48 alone or with imipenem+AVR-48. ** p < 0.01; *** p < 0.001. Data are expressed as mean ± SEM. For statistical analysis, student’s unpaired t-test and one-way analysis of variance (ANOVA) followed by Tukey post hoc analysis were used. CLP: cecal ligation and puncture.