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Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

39237657
71659
10.1038/s41598-024-71659-x
Article
Impact of time intervals on drug efficacy and phenotypic outcomes in acute respiratory distress syndrome in mice
Paris-Robidas Sarah 1
Bolduc Isabelle 1
Lapointe Vanessa 1
Galimi Julia 1
Lemieux Philippe 1
Huppé Carole-Ann carole.ann-huppe@tbt.qc.ca

1
Couture Frédéric frederic.couture@tbt.qc.ca

123
1 TransBIOTech, Lévis, QC G6V 6Z3 Canada
2 https://ror.org/04sjchr03 grid.23856.3a 0000 0004 1936 8390 Nutraceuticals and Functional Foods Institute (INAF), Université Laval, Québec City, QC G1K 7P4 Canada
3 https://ror.org/05ghbjx71 grid.420763.4 0000 0004 4686 6563 Centre Intégré de Santé Et de Services Sociaux de Chaudière-Appalaches, Lévis, QC G6E 3E2 Canada
5 9 2024
5 9 2024
2024
14 2076812 4 2024
29 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Acute respiratory distress syndrome is a severe lung condition resulting from various causes, with life-threatening consequences that necessitate intensive care. The phenomenon can be modeled in preclinical models, notably through the use of lipopolysaccharide (LPS) instillation in mice. The phenotype induced closely recapitulates the human syndrome, including pulmonary edema, leukocyte infiltration, acute inflammation, impaired pulmonary function, and histological damage. However, the experimental designs using LPS instillations are extremely diverse in the literature. This highly complicates the interpretation of the induced phenotype chronology for future study design and hinders the proper identification of the optimal time frame to assess different readouts. Therefore, the definition of the treatment window in relation to the beginning of the disease onset also presents a significant challenge to address questions or test compound efficacy. In this context, the temporality of the different readouts usually measured in the model was evaluated in both normal and neutrophil-depleted male C57bl/6 mice using LPS-induction to assess the best window for proper readout evaluation with an optimal dynamic response range. Ventilation parameters were evaluated by whole-body plethysmography and neutrophil recruitment were evaluated in bronchoalveolar lavage fluids and in lung tissues directly. Imaging evaluation of myeloperoxidase along with activity in lung lysates and fluids were compared, along with inflammatory cytokines and lung extravasation by enzyme-linked immunoassays. Moreover, dexamethasone, the gold standard positive control in this model, was also administered at different times before and after phenotype induction to assess how kinetics affected each parameter. Overall, our data demonstrate that each readout evaluated in this study has a singular kinetic and highlights the key importance of the timing between ARDS phenotype and treatment administration and/or analysis. These findings also strongly suggest that analyzes, both in-life and post-mortem should be conducted at multiple time points to properly capture the dynamic phenotype of the LPS-ARDS model and response to treatment.

Keywords

Acute respiratory distress syndrome model
Acute lung injury
Myeloperoxidase
Dexamethasone
Murine model
Plethysmography
Subject terms

Innate immunity
Acute inflammation
Mouse
Acute inflammation
Fonds de Recherche du Québec- Nature et Technologies291453 Couture Frédéric http://dx.doi.org/10.13039/501100014636 Ministère de l'Éducation et de l'Enseignement supérieur 13380 Couture Frédéric issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Acute respiratory distress syndrome (ARDS) is a life-threatening condition that leads to an increase in pulmonary vascular leakage leading to respiratory failure. The most common cause of ARDS is pulmonary infection, but it can also result from systemic inflammation or traumatic events such as aspiration of gastric content or pancreatitis1. These triggers induce damage- or pathogen-associated molecular patterns, leading to the massive release of cytokines and chemokines and the recruitment of neutrophils, which are usually almost absent from the alveolar space1. ARDS is associated with a high mortality rate in hospitalized patients, reaching 37% in those diagnosed using the Berlin criteria2. Even though clinical trials have been conducted in an attempt to identify therapeutic targets for ARDS, no curative treatment has achieved a positive outcome3. Management of symptoms using ventilation or more controversial treatments such as extracorporeal membrane oxygenation or corticosteroids are the only options available, emphasizing the need to discover new therapeutic targets3.

Preclinical models recapitulating the key clinical features of ARDS used to test compounds or hypotheses mostly rely on the use of a lipopolysaccharide (LPS)—induced lung acute inflammatory response and injury models. This model, which uses a single administration of LPS to the lungs, either intranasally or intratracheally, recapitulates key aspects of human ARDS such as neutrophil infiltration, lung damage, capillary barrier permeation, inflammatory response, and laborious breathing4. This model is widely used across laboratories and research groups to address different aspects of ARDS and to test the efficacy of various compounds in a preclinical context5–7. Although the model is well acknowledged in the literature, there are almost as many experimental designs as there are scientific groups using the model, which often translates into distinct, or even diverging, conclusions. Among the most common variables across experimental designs are the duration of the model, the time at which the readouts are evaluated, and the timing between treatment administration relative to LPS administration.

To address these aspects, we conducted a longitudinal analysis of ARDS phenotype evolution after LPS administration with multiple sacrifice time points and longitudinal analysis of lung function using whole-body plethysmography to evaluate the optimal timing for the different endpoint associated with this model. Neutrophil-depleted mice using anti-Gr1 (Ly6G/Ly6C antibody) were used as a control for neutrophil-associated readouts, which are pillars in this model. Subsequently, using the optimal time point to evaluate most of the key aspects of this model, we further assessed the effect of timing relative to LPS administration with dexamethasone, which is the typical pharmacological positive control in this model. Dexamethasone efficacy was evaluated both systemically, using intraperitoneal (IP) administration, and locally, by intranasal (IN) instillation. LPS was delivered by intranasal instillation since this route is known to yield more consistent results compared to intratracheal delivery8.

Our data demonstrate that key aspects associated with the ARDS phenotype follow unique kinetic patterns, underscoring the critical importance of timing between ARDS phenotype onset and treatment administration or analysis. To accurately capture the dynamic nature of the LPS-induced ARDS mouse model, the timing of analyses and/or treatment administration must be carefully considered.

Results

Kinetic of lung inflammation, damage and impact of respiratory functions displays pivotal dynamic in modelized ARDS phenotypes

To address the time dependence of ARDS-associated phenotype manifestation relative to the initial LPS administration, mice were divided in different subgroups with distinct time of sacrifice and pulmonary function evaluation to closely examine the onset (and offset) of the different readouts. To distinguish the parameters that could be attributed to the direct presence of neutrophils, neutrophil-depleted animals treated with Ly6G/C antibodies were evaluated alongside. Neutrophil depletion has been confirmed by neutrophil hematological counts in the blood at euthanasia (Supplementary Fig. 1).

Early neutrophil infiltration in the lung is one of the hallmarks of ARDS9. The direct neutrophil frequency and the myeloperoxidase (MPO) activity were evaluated at different time points following LPS administration in both the lungs and bronchoalveolar lavage fluid (BALF; Fig. 1A). The percentage of neutrophils in the lung and in the BALF started to significantly increase as early as 4 h after LPS exposure, with a peak between 16 and 24 h (Fig. 1B). Cytometry-based neutrophil frequency in total lung tissue dispersion remained high even after 96 h, while the neutrophils in the BALF decreased with time based on cytospined samples and BALF MPO analyses (Fig. 1C, D). The MPO activity in the lung tissue was significantly increased as early as 4 h and decreased to levels similar to control animals at 96 h after LPS exposure (Fig. 1A). In the BALF, the MPO activity was significantly higher compared to saline after 16 and 24 h and reached a maximum at 48 h after LPS exposure before returning to normal after 96 h (Fig. 1C). The MPO activity in BALF and lung lysates and neutrophil infiltration in the lung remained low in the group treated with the anti-Ly6G/C (Fig. 1A-C). Even though the increase of both neutrophils and MPO activity was observed in the lungs and BALFs, the onset of neutrophil infiltration in lung tissues was faster compared to the BALF. Still, the increase compared to the baseline levels was more pronounced, time-dependent and less variable when using BALF MPO activity, which peaked at 48 h.Fig. 1 Kinetic of Myeloperoxidase Activity in Lungs of ARDS Mice after LPS-Administration (A) MPO activity in whole lung lysates determined by ODA-biochemical assay, (B) neutrophils proportion in lungs determined by flow cytometry analysis, (C) MPO activity in BALF determined by ODA-biochemical assay, (D) neutrophils proportion in BALF determined by manual counting of Giemsa-stained cytospined samples, (E) in vivo thoracic MPO activity determined by CRET-imaging. (F) Density of MPO + cells per mm2 in lung sections determined by quantitative analysis of IHC-stained sections. Representative IHC are displayed in panel G. Data are means ± SEM with (n = 9 for saline and 6/time point for LPS and anti-Ly6G/C + LPS). Scale-bar = 25 µm. *Indicates P-value ≤ 0.05, **0.01, ***0.001 and ****0.0001 compared to saline group from a two-way ANOVA. BAL: bronchoalveolar lavage, MPO: myeloperoxidase.

MPO activity can be assessed in a non-invasive manner using in vivo imaging technique through the use of chemiluminescent resonance energy transfer (CRET) imaging. Four (4) and 24 h after LPS administration, mice were subjected to full-body imaging following intravenous injection of luminol along with near-infrared quantum dot (QD) nanoparticles to generate MPO-induced UV light, which is converted by the QDs to image CRET-generated infrared-shifted luminescence10. The measured thorax photon flux was significantly increased after 24 h in mice exposed to LPS compared to saline or mice treated with the anti-Ly6G/Ly6C antibody (Fig. 1E). Although the in vivo imaging could be used to evaluate neutrophil content in the lung tissues non-invasively, it displayed low sensitivity, as depicted by the lack of signal increase after 4 h of induction and only a modest increase of the signal after 24 h when compared to controls. Moreover, the ratio between signal and background did not appear large enough to justify the use of this approach.

Analysis of lung sections for the presence of MPO-positive cells by IHC (Fig. 1F-G) in lungs mirrored the results obtained in lungs by flow cytometry (Fig. 1B) and from BALF (Fig. 1D). The number of MPO-positive cells per mm2 was higher by ~ 5-folds at the 4 and the 24 h time points in mice exposed to LPS compared to saline. Finally, as seen with the enzymatic assay performed with lung tissues, the MPO+ cells were also present in the lungs of animals treated with anti-Ly6G/Ly6C. However, as observed in Fig. 1A, this effect occurs at a lower extent compared to the LPS only group.

This LPS-induced model recapitulates key features of ARDS, notably the impairment of respiratory functions due to the inflammatory response and associated vascular leakage which results in increased lung stiffness, diminishing their compliance and increasing the respiratory effort required for breathing. The use of whole-body plethysmography, a non-invasive ventilation assessment technique, allows repeated data collection in non-restrained animals11. The animals from the different groups were evaluated prior to LPS administration and at different time points to assess the evolution of the respiratory phenotype. Figure 2 presents the changes from baseline values using the different parameters evaluated at 1, 3, 6, 16, 24, 48, and 96 h after LPS administration.Fig. 2 Kinetic of Pulmonary Function in ARDS Mice after LPS-Administration Relative changes from each individual mouse value prior to LPS administration for (A) Penh, (B) Ti/Te ratios, as determined by whole-body plethysmography. Additional parameters can be found in Supplementary Fig. 2. Data are means ± SEM with (n = 9 for saline and 6/time point for LPS and anti-Ly6G/C + LPS). *Indicates P-value ≤ 0.05, **0.01, ***0.001 and ****0.0001 compared to saline group whereas #Indicates P-value ≤ 0.05, ##0.01, ###0.001 and ####0.0001 compared to LPS group from a two-way ANOVA. Te: expiratory time, Ti: inspiratory time.

LPS exposure significantly increased the enhanced pause (Penh) 1 h post-LPS instillation while reaching a sixfold increase from baseline values 6 h after LPS administration (Fig. 2A). The neutrophil depletion significantly delayed the onset of the pulmonary obstruction phenotype until 16 h post-LPS administration as shown by the shift in the Penh values peak. The LPS instillation resulted in a Penh onset at approximately 6 h with persisting elevation until ~ 24 h post-LPS.

The ratio between the inspiratory time on the expiratory time (Ti/Te) was reduced by LPS with a rapid reduction starting after 1 h and peaking after 16 h following instillation (Fig. 2B). Both Penh and Ti/Te alterations were alleviated 48 h post-LPS and completely resolved after 96 h. Animals pretreated with the anti-Ly6G/C antibody showed a generally similar pattern in terms of changes in pulmonary functions, as neutrophil-depleted mice showed reduced phenotypes that were both delayed in time based on the pulmonary functions evaluated.

Other plethysmography parameters were also evaluated following LPS exposure, notably the mid-expiratory flow (EF50), breathing frequency, tidal volumes and minute ventilation. These data are presented in Supplementary Fig. 2 and only displayed mild alterations that were mostly physiologically insignificant. Of all the plethysmography-based measurements performed, Penh appeared as the best indicator of the induced phenotype as it had increases of up to 6-folds compared to baseline values.

To evaluate the degree of both inflammation and the permeation of the alveolar-capillary barrier, the concentrations of TNF-α, IL-1β, albumin and total protein in the BALF were quantified at the different endpoints. LPS exposure resulted in a rapid and transient increase of TNF-α levels in the BALF. TNF-α levels reached ~ 20 times the basal levels only after 4 h following LPS administration but rapidly returned to levels similar to the baseline (only ~ 5 folds remaining after 16 h and in the same range as control after 24 h; Fig. 3A). The increases in the concentrations of IL-1β in BALF were already observable 4 h after LPS instillation and the rise persisted up to 48 h afterward (Fig. 3B). Neutrophil depletion did not impact the TNF-α nor the IL-1β release in the lung. The albumin concentrations peaked at 16 h post-LPS, and decreased afterward but remained significantly different from the saline control for the rest of the study (Fig. 3C). The total protein in the BALF was also increased 16 h post-LPS exposure, and the concentration was still higher than saline control 96 h post-LPS (Fig. 3D). Albumin is likely a more accurate measure of extravasation than total proteins, which explains the disparity between the two readouts.Fig. 3 Evolution of IL-1ꞵ and Albumin in Bronchoalveolar Lavages Following LPS Administration Bronchoalveolar fluids (BALF) concentrations of (A) TNFα, (B) IL-1ꞵ, (C) albumin, determined by ELISA and (D) total protein, determined by BCA protein assay at the indicated time point following LPS administration. Data are means ± SEM with (n = 9 for saline and 6/time point for LPS and anti-Ly6G/C + LPS). *Indicates P-value ≤ 0.05, **0.01, ***0.001 and ****0.0001 compared to saline group whereas ##Indicates P-value ≤ 0.01, compared to LPS group from a two-way ANOVA.

As a result of the local inflammatory response and recruitment of immune cells in the lung, tissue alterations were evaluated based on acute inflammation infiltration and mononuclear cell aggregates (see Table 1 in Supplementary Material) on fixed lung sections. LPS exposure increased the overall histological scores mostly because of an increase in acute inflammation infiltration (Fig. 4A). As shown in Fig. 4B-C immune cell infiltration generally increased over time to reach a peak at 96 h post-LPS. The treatment with anti-Ly6G/C antibodies alleviated the tissue damage with significant differences with LPS animals across the different time points evaluated.Fig. 4 Kinetic of Lungs Damage and Inflammation after LPS Administration (A) Histological scores of mice lungs at the indicated time point determined by H&E sections scoring. (B) Representative H&E from reference (saline instillation instead of LPS) animals. (C) Representative H&E from animals receiving LPS and (C) antiLy6G/C + LPS at the indicated time points. Data are means ± SEM with (n = 9 for saline and 6/time point for LPS and anti-Ly6G/C + LPS). *Indicates P-value ≤ 0.05, **0.01 and ****0.0001 compared to saline group whereas #Indicates P-value ≤ 0.05, ##0.01, compared to LPS group from a two-way ANOVA.

The timing of corticosteroid treatment with dexamethasone is crucial to properly hinder ARDS-related phenotypic features

Considering the sum of the entire data and the readouts kinetics (Figs. 1–4), the 24 h post-LPS administration timepoint was selected to evaluate treatment efficacy after a single dosing. Although a few readouts peaked either before or after this time point, the response window at 24 h allowed a clear picture of ARDS hallmark features including inflammation, histological damage, neutrophil recruitment, pulmonary edema, and labored breathing. However, the dynamics of this induction raised the question of whether the timing of treatment administration was equally crucial? Considering the succession of steps that culminate in ARDS, dexamethasone, a corticosteroid, was chosen as it is the most recurrently used positive control in this model. To investigate the impact of different timing relative to the moment of LPS administration on readouts, various administration times were explored; i.e., 24, 2 or 1 h before as well as 2 h and 1 h after LPS administration. Dexamethasone was administered either systemically (intraperitoneally), or locally (intranasally), to evaluate the differential efficacy it could yield depending on the route of administration.

The neutrophil infiltration was evaluated by measuring the frequency of neutrophils in BALF and the MPO activity in BALF and lung tissue. The percentage of neutrophils was almost null in control mice while reaching 80% of total cells after LPS exposure. However, dexamethasone treatments whether administered systemically or locally, did not significantly reduce the neutrophil percentage (Fig. 5A-B). The MPO activity in BALF was increased following LPS exposure (~ 200 U/mg) in LPS compared to 55 U/mg in saline). Nonetheless, administration of dexamethasone 2 h before LPS by IP injection or IN instillation 2 h pre- and 1 h post-LPS significantly reduced the MPO activity to ~ 125, 110, and 135 U/mg, respectively (Fig. 5C-D). In pulmonary tissues, the number of MPO-positive cells was quantified following an IHC staining, and the number of MPO + cells tended to increase following LPS exposure. Dexamethasone treatment, either 2 or 1 h post-LPS administration by IP injection and 2 h before LPS by IN instillation, decreased the number of MPO + cells to the same level as saline-induced animals (Fig. 5E-F).Fig. 5 Distinctive Efficacy of Dexamethasone at Different Timing and Using Different Routes of Administration on Neutrophil-Related Readouts (A) Neutrophil proportions in BALF from mice treated intraperitoneally or (B) intranasally at the indicated time points. (C) MPO activity in BALF determined by ODA-biochemical assay on BALF from mice treated intraperitoneally or (D) intranasally at the indicated time points. (F) Density of MPO + cells per mm2 in lung sections determined by quantitative analysis of IHC-stained sections. Representative IHC are displayed in panel G. Data are means ± SEM with (n = 9 for saline and LPS and 5–6 for Dexamethasone treated groups). Scale-bar = 25 µm. *Indicates P-value ≤ 0.05, **0.01 and ****0.0001 compared to saline group from a one-way ANOVA. BAL: bronchoalveolar lavage, EF50 midexpiratory flow, IN intranasal, IP intraperitoneal, MPO myeloperoxidase, Te expiratory time, Ti inspiratory time Tx treatment.

The respiratory parameters were measured using the whole-body plethysmography technique, this time solely at 24 h post-LPS administration. LPS exposure increased the Penh by ~ 160% compared to the baseline, while it remained unchanged in mice exposed to saline only. Administration of dexamethasone by IP injection 2 h or 1 h before LPS prevented the associated increase in Penh values. On the other hand, administration by intranasal delivery did not impact Penh values (Fig. 6A-B).Fig. 6 Efficacy of Dexamethasone at Different Timing and Using Different Routes of Administration on Respiratory Parameters Relative changes from each individual mouse value prior to LPS instillation for (A-B) Penh, (C-D) EF50, (E–F) breathing frequency, and (G-H) Minute ventilation 24 h after LPS when treated at the indicated time before or after LPS either intraperitoneally (IP) or intranasally (IN) were determined by whole body plethysmography. Data are means ± SEM with (n = 9 for saline and LPS and 5–6 for Dexamethasone treated groups). *Indicates P-value ≤ 0.05, **0.01, ***0.001 and ****0.0001 compared to saline group from a one-way ANOVA. EF50: midexpiratory flow, Tx: treatment. Additional parameters can be found in Supplementary Fig. 3.

In this study, LPS exposure decreased the EF50 at 24 h compared to the saline control group. Typically, a decrease in EF50 has been associated with bronchoconstriction as seen in experimental asthma12. Treatment with dexamethasone by IP injection 2 h before LPS administration was the best condition for restoring normal EF50 compared to saline (Fig. 6C-D). LPS decreased the overall breathing frequency (Fig. 6E) and for this parameter, dexamethasone administered either IP 2 or 1 h before or after LPS restored the breathing frequency to the normal range. However, when administered intranasally, no effect was observed (Fig. 6F). Finally, the observed minute ventilation parameters showed reduced values induced by LPS. This reduction was mitigated by IP administration of dexamethasone when administered 2 h or 1 h before or after LPS exposure, but not 24 h before (Fig. 6G). Intranasal administration did not provide any effect either (Fig. 6H). Ti/Te ratios and tidal volumes remained unaffected and are reported in Supplementary Fig. 3.

Following the evaluation of the impact of dexamethasone treatment on ventilatory parameters, the anti-inflammatory effects and preservation of the alveolar-capillary barrier functions were evaluated by measuring IL-1β and albumin concentrations in the BALF (Fig. 7). LPS-challenge readily increased the concentrations of IL-1β in the BALF, and IP dexamethasone treatment either 2 or 1 h before or after the LPS administration prevented this cytokine secretion (Fig. 7A). The 24 h pretreatment did not result in any impact on this parameter. Interestingly, intranasal treatment with dexamethasone displayed an anti-inflammatory effect when administered 2 h before and reduced the concentration of IL-1β, but when given 1 h after the LPS, the observed effects were weaker (Fig. 7B).Fig. 7 Efficacy of Dexamethasone at Different Timing and Using Different Routes of Administration on IL-1ꞵ and Albumin in Bronchoalveolar Lavages Concentrations of (A-B) IL-1ꞵ, (C-D) albumin and (E–F) total proteins in BALF in mice 24 h after LPS-instillation when treated intraperitoneally (IP) or intranasally (IN) at the indicated time points before or after LPS. Data are means ± SEM with (n = 9 for saline and LPS and 5–6 for Dexamethasone treated groups). *Indicates P-value ≤ 0.05, **0.01 and ***0.001 compared to saline group from a one-way ANOVA. IN: intranasal, IP intraperitoneal, Tx treatment.

The concentrations of albumin were measured as an indicator of LPS-induced pulmonary leakage. LPS-challenge resulted in an increased albumin concentration in the BALF. Only treatment with dexamethasone IP 1 h, before LPS tended to reduce the albumin leakage (Fig. 7C). Furthermore, no efficacy was observed when administered intranasally.

Finally, the efficacy of dexamethasone to protect the lungs from LPS-induced tissue damage was evaluated based on the histological evaluation of acute immune cell infiltrate and mononuclear cell aggregates. LPS increased the overall histology scores in the lung from ~ 1.0 to ~ 2.3, but none of the tested treatment regimens with dexamethasone reduced the histological damage observed (Fig. 8).Fig. 8 Tissue Damage Evaluation Following Treatment with Dexamethasone Using Different Treatment Timing and Route of Administration (A) Histological scores of mice lungs 24 h after LPS instillation when treated intraperitoneally at the indicated time points before or after LPS. (B) Histological scores of mice lungs 24 h after LPS instillation when treated intranasally at the indicated time points before or after LPS. Data are means ± SEM with (n = 9 for saline and LPS and 5–6 for Dexamethasone treated groups) . Scale-bar = 25 µm ****Indicates P-value ≤ 0.0001 compared to saline group from a one-way ANOVA. IN intranasal, IP intraperitoneal, Tx treatment.

Discussion

In human, key parameters are necessary to diagnose ARDS13. Patients must display failure of the respiratory system occurring within one week of a recognized injury or the emergence and/or deterioration of respiratory symptoms. This respiratory failure must also not be entirely attributed to cardiac function or volume overload, and the oxygenation defect must reach a significant level as assessed by the acute onset of hypoxaemia (SpO2/FiO2 < 315 mm Hg). ARDS is most commonly observed in the context of pneumonia (either bacterial or viral in most cases), non-pulmonary sepsis or following trauma14.

The pathogenesis-associated mechanisms leading to the manifestations of ARDS rely on the lung endothelium, which exhibits increased permeability to fluids and proteins. This results in edema within the lung interstitial space, accompanied by the accumulation of red blood cells in the alveolar space15,16. This accentuated permeation leads to a mismatch in ventilation and perfusion and intrapulmonary shunting that impairs CO2 excretion contributing to respiratory failure. Endothelial cell disruption, which can be caused by microorganisms or their components like LPS or toxins following exhaustive endothelial cell activation and chemokine secretion. This then triggers immune cells and platelets accumulation in the lung vasculature17. These successive events result in alveolar haemorrhage and atelectasis. Several studies have highlighted that these damages are exacerbated by platelet activation and neutrophil recruitment and degranulation17,18. Through the release of proteases and oxidizing enzymes such as elastase and MPO, the lungs are then injured. In human cases, tissue injuries are worsened by mechanical ventilation, which exerts a strong pressure on the already inflamed tissues.

The LPS-induced ARDS model in mice, also sometimes referred to as acute lung injury (ALI), has the advantages of recapitulating most of these key features over a rather short period of time following induction (Figs. 1–2-3–4). However, the terminology ARDS and ALI are also used to refer to animal models induced using other triggers, notably live bacteria, bleomycin and oleic acid, to name a few5. The LPS-induced model is simple, involving a neutrophil response to the lungs directly without the requirement of live bacteria (and corresponding biosafety level). Within the first 4 h after the induction in mice, it is already possible to depict neutrophil accumulation both within the lung tissues (Fig. 1A-B & F) but also in the alveolar lavages (Fig. 1D)19. Pulmonary leakage, as depicted by the elevation of protein as well as albumin concentrations in the BALF, occurs within the first 16 h after induction (Fig. 3C-D) and reflects on the pulmonary functions. As quickly as 1 h after LPS administration, the Penh values measured by whole-body plethysmography rise and remained high for more than 3 days (Fig. 2A). The results obtained in the neutrophil-depleted mice really highlighted the importance of timing since the depletion delayed the onset of respiratory changes and the impact of neutrophils on pulmonary functions could have been missed if only the wrong time point had been considered. The Penh parameter is a controversial parameter since experts in the field showed it did not correlate with an increased airway resistance as determined using invasive measurement technique20. However, this parameter has been used in multiple in vivo preclinical studies in mice and there is a dose–response association between elevated Penh and viral dose in a mouse model of SARS-CoV-2 infection21. In the current study, the Penh was used as a nonspecific indicator of breathing alteration or airway obstruction as a result of diminished respiratory compliance and not as a measure of airway resistance. The changes in ventilatory parameters (Fig. 2A) correspond with the recruitment of neutrophils in the lung tissues (Fig. 1A-B), which occurs prior to their migration into the BALF. This aligns with the observation that transepithelial migration of cells to the alveolar space is delayed compared to tissue recruitment. Their migration in the fluid was mostly depicted after 16 h following LPS, which is coherent with former observations showing a peak 24 h after LPS challenge22. Damages to the lung structures were mostly depicted 48–96 h post-LPS induction (Fig. 4C). Overall, these observations, but most precisely their exact dynamic over time, allowed a clear depiction of the best window for readout evaluation, but also for intervention. Figure 9 present the chronology of these events as per these different readout measurements.Fig. 9 Temporal Dynamics of Acute Respiratory Distress Syndrome in Mice Following LPS Administration Schematic representation of the interaction between neutrophil recruitment and pulmonary leakage following the initial secretion of TNFα and subsequent IL-1β, resulting in the associated respiratory efforts and lung damage.

The analysis performed on various ventilation parameters using whole-body plethysmography revealed that the observed changes varied depending on the timing of comparative readings. It is crucial to note that these differences may account for variations between experiments or with previous literature, as different time points are often utilized for assessment.

The evaluation of the neutrophil-derived MPO enzyme is well recognized in the literature as a key readout since neutrophil recruitment plays a critical role in the pathogenesis induced by this model23,24. Using an antibody-mediated depletion strategy with the anti-Ly6G/C administration, we evaluated the specific contribution of neutrophils in the different readouts following LPS-administration (Figs. 1–4). We chose to use the RB6-8C5 clone targeting Gr1, which was reported to provide a stronger and longer-lasting neutrophil depletion prior to rebounding25. Although targeting Gr1 can result in some unspecific cell depletion, as monocytes are also Gr1+ cells, blood analysis performed to evaluate both cell types during the course of the study showed that compared to LPS and control mice, neutrophils were significantly diminished, not monocytes (Supplementary Fig. 1B)26. This strategy is also more specific than the use of cyclophosphamide to induce neutropenia as previously reported27. To measure MPO activity, lung tissue samples need to be homogenized and further used in enzyme kinetic assays. Technically, measuring the activity in lung tissues represents a certain challenge since the enzyme activity is rather labile over time. In our experience, it needs to be assessed either on fresh tissues or within the following 48 h of sample freezing at − 80 °C to avoid loss of activity. Interestingly, the activity in lung tissues and in BALF was compared and the amplitude of induction of MPO activity (saline compared to LPS-induced) in the BALF showed a higher range of induction (up to tenfold for BALF compared to sixfold change in lungs; Fig. 1A-C). Activity measurement in BALF is thus reliable, easier and faster than in tissues, making it a method of choice to assess neutrophil recruitment. Measurement of MPO activity in vivo using CRET-imaging was also performed. Although it was possible to distinguish elevation in the neutrophil-derived MPO in the thoracic cavity of the LPS-induced mice compared to saline-induced group and the neutrophil-depleted animals (Fig. 1E), the fold-change remained modest, and the obtained signals were too low to represent a valuable readout compared to enzymatic assay-derived data28,29. This is most likely attributable to the tissue depth as the technique displayed much higher signals when conducted on skin located lesions30.

Based on our longitudinal observations, it is clearly important to consider the temporal onset of different pathological manifestations and their incidence on the readouts when assessing the efficacy of given treatments to impact the ARDS-related phenotypes (Fig. 9). For example, the evaluation of inflammatory response by measuring IL-1ꞵ should only be done within the first 4–48 h (Fig. 3B) whereas lung extravasation can persist for 4 days (Fig. 3C-D). Although TNF-α elevation is significant, it is also highly transient (Fig. 3A), making it challenging to identify suitable windows to assess compound efficacy using this cytokine. As highlighted by the disparity between the albumin and the total protein concentrations measured in the BALF, albumin levels likely reflect the peak of bloodstream protein leakage, whereas total protein measurement could be considered as a gross reflection of epithelial cell response and cell recruitment to the lungs. Only a few studies have compared total protein content and albumin in BALF8,31. According to those publications both readouts show evidence of extravasation. However, based on our data (Fig. 3C-D), measuring of albumin could be advantageous, as it shows a larger dynamic range for response evaluation. Following the observation of neutrophil recruitment, histological damage persisted longer due to the prolonged presence of inflammatory mediators and neutrophils, as indicated by the diminished phenotype in anti-Ly6G/C treated animals (Fig. 4). The lack of effect of the neutrophil depletion on TNF-α and IL-1ꞵ secretion in the BALF is consistent with the understanding that these cytokines are respectively secreted by macrophages and type I and II alveolar cells in response to LPS, rather than by neutrophils32.

The administration of corticosteroids, such as dexamethasone, to ARDS patients has been a topic of debate for several years. Cumulative evidence now points toward beneficial effects, often reported in terms of the duration of mechanical ventilation or patient mortality33. However, the range of ARDS-causing agents is broad, and not all conditions may respond similarly to treatments. The recent SARS-CoV-2 pandemic has provided large sample size analyses, with several reports indicating favorable outcomes for ARDS patients receiving corticosteroids compared to standard care34,35. Dexamethasone is used as a positive control or adjunct therapy in a numerous studies using the LPS-induced ARDS model in mice, as it attenuates the induced phenotype depending on the readout used to assess efficacy5. When evaluating the timing of dexamethasone administration in relation to its efficacy after a single dose, our results suggest that it should be administered before the LPS induction to achieve the highest response, ideally 1–2 h before.

Prophylactic administration of dexamethasone is expected to prevent the onset of inflammation by modulating the immune response and decreasing the production of pro-inflammatory cytokines. Regardless of the route of administration, diminished MPO activity (Fig. 5A-B) and albumin leakage in the BALF (Fig. 7C-D) were observed only following a preventive treatment. This is consistent with published evidence that prophylactic treatments, particularly as a single dose like in the present study, are more efficient36. Interestingly, systemic administration either before or after LPS-induction blocked IL-1ꞵ secretion in the BALF (Fig. 7A), whereas this was only observed in prophylactically treated animals when using the intranasal route (Fig. 7B). Only systemic administration of dexamethasone resulted in a beneficial effect on ventilation parameters, particularly Penh (Fig. 6). These observations correlate directly with the observed neutrophils in the lung tissues (Fig. 5E-G). This likely reflects the pharmacokinetic/pharmacodynamic relationship of dexamethasone. Pharmacokinetic data from published studies indicate that intranasal dexamethasone achieves approximately 80% bioavailability and circulating levels are comparable to intravenous delivery37,38. In our study, we administered a dose intranasally 10 times lower than the intraperitoneal doses (0.5 vs 5 mg/kg), taking into consideration that the goal was to assess direct lung effects rather than systemic exposure. This lower dosage was reported to be effective when administered intranasally in a model of tuberculosis39. Dexamethasone has a half-life of ~ 2.3 h in C57Bl/6 mice and ~ 2.6 h in rats, with Cmax of ~ 8 h after IP injection, which likely explains the need to administer the compound not long before or after the LPS administration to obtain maximal efficacy during the onset of inflammatory response40,41. In the current study, the intraperitoneal route of administration was favored to replicate what is most commonly done in the literature42,43. This administration route is often favored because it is easier from a technical point of view and because dexamethasone penetrates tissues rapidly yielding a large distribution volume40. Dexamethasone can act directly by suppressing cytokine expression as well as Toll-Like Receptor (TLR) activity, thereby preventing the development of the ARDS-associated phenotype39,44. Published reports showed that histological damage was stronger after 72 h and that dexamethasone efficacy was more easily observed at this time point compared to earlier time points such as 24 h after induction45,46. This may explain why we did not observe much protective effect against histological damage (Fig. 8). Moreover, some published studies instead used repeated administrations of dexamethasone in addition to the first administration close to the LPS-instillation and showed clearer reductions of LPS-induced histological damage47. In the present study, we only focused our analysis on the effects of a single administration which is also consistent with the short time interval selected for readout assessment.

However, data from dexamethasone administered 1 h after LPS show a clear reduction of histological damage when observed 7 h after induction but only a mild effect on inflammation markers such as IL-1ꞵ48. Still, because of the various ways the model is induced across different research groups, the efficacy varies significantly. Some groups are notably administering LPS more than once, sometimes intraperitoneally instead of intranasally, and sometimes with several administrations of compounds preceding the induction49–52. This may notably explain the disparity in the degree of induction and response to different agents in the literature, but it also highlights the necessity to provide a detailed method description. It is also important to note that the type of LPS used, the source organism serotypes (e.g., E. coli O111 in our case), and the endotoxin content are likely to impact the resulting phenotype. These factors can affect how the TLR, especially TLR4, is modulated and influence the required dosage to elicit the same degree of lung inflammation53 It is also important to note that data obtained in the rat LPS-induced ARDS model differs slightly from those obtained in mice and from mice directly challenged with microorganisms54,55.

In conclusion, these data support the necessity of fine-tuning the timing of LPS induction, treatment administration, as well as the appropriate timeframe for readout assessment in this preclinical ARDS model. In our current study aiming to evaluate the efficacy of a single dose of a pharmacological agent, using dexamethasone, we identified the 16–24 h post-LPS timeframe as the optimal window to examine the effect on pulmonary function, neutrophil recruitment and pulmonary leakage. The evaluation of histological damage should be conducted at a longer time point, ideally 4 days after the LPS instillation. Depending on the duration of the compounds’ effects, multiple administrations may be needed to address histological damage over time. However, a drug that sufficiently mitigates the initial local inflammatory response is likely to reduce the histological damage, as observed with neutrophil depletion (Fig. 4).

Material & methods.

Animals

TransBIOTech animal care facility is accredited by the Canadian Council on Animal Care (CCAC). This study (#001–22) was approved by the Cégep de Lévis Animal Care Committee and performed in compliance with CACC standards and regulations governing the use of animals for research. The experimental design of this study (research question, design features, readouts, and analysis plan) was planned prior to being conducted. No specific criteria to include or exclude data or animals were used. All data and animals were used in these studies. This study was conducted in accordance with the ARRIVE guidelines.

Male C57BL/6 mice, 6–8 weeks of age (Charles Rivers Laboratories, St-Constant, QC) were used for this study. A total of 114 mice were used in the study. All groups were composed of 5–6 mice based on the usual standard deviation and variability in this model. Control groups (i.e., PBS and LPS alone) were performed alongside other conditions and were combined to yield n = 6–9/group. The numbers are indicated below each graph. Following arrival, animals were subjected to an acclimation period of 7 days before the beginning of the study.

The mice were housed in groups under standardized environmental conditions in auto-ventilated cages (75 air changes per hour). Tap water and standard certified commercial rodent diet (Envigo 2018) enriched with Enviro-dri (Shepherd Specialty Papers, USA), and Nestlet (Ancare Corporation, USA) were provided ad libitum except during designated procedures requiring the handling of animals outside of their housing cages. Each cage was identified for the corresponding group, indicating the treatment and the identity of the animals housed in the cage. The housing room was maintained under controlled conditions (temperature: 21.0 ± 1 °C, relative humidity: 40 ± 10%, 12 air changes per hour and 12-h light/dark cycle). All mice were assigned into groups based solely on body weight to ensure homogeneity. Standard animal care was provided to animals. No mice had to be terminated after reaching humane endpoints (body weight loss, sign of pain or distress) in this study and no unexpected events were observed. Studies were not conducted blindly.

ARDS induction and treatments

ARDS was induced by intranasal LPS (100 µg; Millipore Sigma, from Escherichia coli O111:B4, #L3012-25MG) instillation using a micropipette. The instillation was performed under isoflurane anesthesia. The total volume (50 µL) was equally administered dropwise between the two nostrils in synchronicity with the animal’s inspiration. In the first set of experiments, 100 µg of anti-Ly6G/Ly6C (RB6-8C5; BioXCell, Lebanon, NH, USA) was administered intraperitoneally to mice 3 days prior to LPS administration to induce neutrophil depletion. For animals terminated more than 24 h after the LPS administration, an additional dose of 100 µg was administered 24 h after the LPS. In the second set of experiments, Dexamethasone (Sigma, #D2915-100 mg) was administered intraperitoneally (5 mg/kg) or intranasally (12.5 µg; or approximately 0.5 mg/kg). Dexamethasone treatments were administered either before (24 h, 2 h, or 1 h) or after (2 h or 1 h) LPS administration. Detailed sample collection procedures are provided in the Supplementary Material.

Chemiluminescence resonance energy transfer (CRET) imaging

For in vivo imaging of MPO activity, the procedure was adapted from Zhang et al.28 and performed at the indicated time point. The detailed procedure is provided in the Supplementary Material.

Bronchoalveolar lavage, histology and immunohistochemistry

The BALF were processed using a Cytospin 4 Cytocentrifuge (Epredia, Waltham MA, USA) and fixed for Wright-Giemsa stained (Hemacolor Rapid staining, Sigma) prior to mounting for manual differential count. Paraffin-embedded tissues were H&E or IHC stained using standard procedures. The detailed sample treatment and scoring procedures are provided in the Supplementary Material. All slides were visualized using a digital slide scanner (PANNORAMIC MIDI II, 3DHistech, Budapest, Hungary).

MPO ODA-biochemical activity assay

For MPO activity measurements on frozen lungs or on BALF, the method already described in Babity et al. was used30. The detailed procedure is provided in the Supplementary Material.

whole-body plethysmography

The mouse respiratory function was evaluated using the vivoFlow whole-body plethysmograph (SCIREQ, Montreal, QC). Before data acquisition, the mice were acclimated to the acquisition chamber twice for 20 min each time. Before treatment or LPS administration, the mice were placed in the acquisition chamber. An acclimation period of 5 min was allowed and respiratory function was recorded for 15 min. Data acquisition was repeated at euthanasia time.

Lungs flow cytometry analysis

The lungs were minced with surgical scissors and digested for flow cytometry staining. Data were acquired on the BD LSR Fortessa X-20 flow cytometer. The flow cytometry data were analyzed using FlowJo software (BD). The detailed procedure is provided in the Supplementary Material.

ELISA

The concentration of TNF-α, IL-1β and albumin in the BALF fluid were evaluated using routine ELISA assays following the procedure recommended by the manufacturer. The detailed procedure is provided in the Supplementary Material.

Statistics

Data were presented using the means ± SEM. All groups shown on a graph were compared to one another, unless indicated otherwise in the figure legends. An experimental unit was always considered to be an individual animal. The figures and statistical analysis were generated using the GraphPad Prism v.10.0.3 software. The statistical significance was evaluated using a two-way ANOVA with a Dunnett’s post hoc test for multiple comparisons when analyzing grouped data. The measurement of respiratory function at different time points was analyzed using a mixed-effect analysis with a Tukey’s post hoc test for multiple comparisons. The statistical significance for the comparison of the different treatment groups was evaluated using a one-way ANOVA with a Dunnett’s post hoc test for multiple comparisons.

Supplementary Information

Supplementary Information.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-71659-x.

Acknowledgements

The authors want to thank the entire TransBIOTech animal care team, particularly Ms Sara Carignan, Ms Gabrielle St-Pierre and Mr Samuel Desbecquets of for their technical assistance as well as Mr Charles-Antoine Fournier for his technical help for flow cytometry analyzes.

Author contributions

IB, JG, VL and PL contributed to the study Investigation and Methodology. FC and CAH contributed to the study Conceptualization FC, CAH and SPR contributed to Formal Analysis and wrote the manuscript FC obtained the Funding All authors reviewed the manuscript.

Funding

This study is supported by the Fonds de Recherche du Québec- Nature et Technologies (FRQ-NT; Grant # 291453) to F.C. The publication of this study is also funded by the Ministère de l’Enseignement Supérieur du Québec through the PADRRC program (Grant #13380).

Data availability

Data are available upon contacting the Corresponding Author.

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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