Safety, tolerability, and biomarkers of the treatment of mice with aerosolized Toll-like receptor ligands

Victoria Y Alfaro,Adam K Walker,Gabriella R Valverde,Burton F Dickey,Lee J Quinton,Scott E Evans,Mark F Munsell,David L Goldblatt,Atul Varadhachary,Robert Dantzer,Brenton L Scott,Michael J Tuvim

doi:10.3389/fphar.2014.00008

Abstract

We have previously discovered a synergistically therapeutic combination of two Toll-like receptor ligands, an oligodeoxynucleotide (ODN) and Pam2CSK4. Aerosolization of these ligands stimulates innate immunity within the lungs to prevent pneumonia from bacterial and viral pathogens. Here we examined the safety and tolerability of this treatment in mice, and characterized the expression of biomarkers of innate immune activation. We found that neutrophils appeared in lung lavage fluid 4 h after treatment, reached a peak at 48 h, and resolved by 7 days. The peak of neutrophil influx was accompanied by a small increase in lung permeability. Despite the abundance of neutrophils in lung lavage fluid, only rare neutrophils were visible histopathologically in the interstitium surrounding bronchi and veins and none were visible in alveolar airspaces. The cytokines interleukin 6 (IL-6), tumour necrosis factor, and Chemokine (C-X-C motif) ligand 2 rose several hundred-fold in lung lavage fluid 4 h after treatment in a dose-dependent and synergistic manner, providing useful biomarkers of lung activation. IL-6 rose fivefold in serum with delayed kinetics compared to its rise in lavage fluid, and might serve as a systemic biomarker of immune activation of the lungs. The dose–response relationship of lavage fluid cytokines was preserved in mice that underwent myeloablative treatment with cytosine arabinoside to model the treatment of hematologic malignancy. There were no overt signs of distress in mice treated with ODN/Pam2CSK4 in doses up to eightfold the therapeutic dose, and no changes in temperature, respiratory rate, or behavioral signs of sickness including sugar water preference, food disappearance, cage exploration or social interaction, though there was a small degree of transient weight loss. We conclude that treatment with aerosolized ODN/Pam2CSK4 is well tolerated in mice, and that innate immune activation of the lungs can be monitored by the measurement of inflammatory cytokines in lung lavage fluid and serum.

Highlights

Infectious pneumonia exacts a high burden of morbidity and mortality worldwide (Mizgerd, 2008; Lozano et al, 2012)
We found that innate defenses of the lungs of mice can be stimulated therapeutically to induce a high level of resistance to microbial infection (Clement et al, Abbreviations: i.p., intraperitoneally; i.v., intravenously; LPS, lipopolysaccharide; ODN, oligodeoxynucleotide; ODN M362, TCG TCG TCG TTC GAA CGA CGT TGA T; O/P, ODN M362 and Pam2CSK4 at a 1:4 molar ratio; Pam2CSK4, 2,3-bis(palmitoyloxy)-2-propyl-Cys-Ser-Lys-Lys-Lys-Lys-OH; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; Serum amyloid A1 (SAA1), serum amyloid A1; TLR, Toll-like receptor. 2008; Tuvim et al, 2009; Evans et al, 2010a,b)
KINETICS OF LUNG INFLAMMATION AFTER A SINGLE O/P EXPOSURE To assess the onset and duration of lung inflammation resulting from a single exposure to 4 ml of aerosolized 1 μM ODN and 4 μM Pam2CSK4, we measured cytokines and leukocytes in lung lavage fluid

Summary

Introduction

Infectious pneumonia exacts a high burden of morbidity and mortality worldwide (Mizgerd, 2008; Lozano et al, 2012). Resistance to infection was accompanied by transient inflammation within the lungs, including an influx of neutrophils and cytokines into lavage fluid (Clement et al, 2008; Tuvim et al, 2009). Treatment appeared to be well tolerated with no apparent adverse behavioral effects, only a minimal rise of cytokines in serum, and resolution of lung inflammation within a week (Clement et al, 2008; Tuvim et al, 2009)

Methods

Results

Discussion

Conclusion