Effects of Intratracheal Mesenchymal Stromal Cell Therapy during Recovery and Resolution after Ventilator-induced Lung Injury

MECHANICAL ventilation is essential to support life in acute respiratory failure, but can worsen lung injury severity.^1,2  Repeated cycles of mechanical stretch result in lung inflammation, injury, and ultimately tissue destruction—this is termed ventilator-induced lung injury (VILI).^3,4  The mechanisms by which mechanical ventilation can worsen the severity of acute lung injury (ALI) and Acute Respiratory Distress Syndrome (ARDS) are clear.⁵  Conversely, it is clear that conventional low stretch mechanical ventilation strategies save lives.^6,7 

Recent studies have generated considerable interest in human mesenchymal stromal cells (MSCs) as a therapeutic option for patients suffering with ALI/ARDS. MSCs attenuate inflammation and lung injury in preclinical ARDS models.^8,9  An important mechanism of action of human MSCs is restoration of alveolar fluid clearance, which has been demonstrated to occur via secretion of keratinocyte growth factor.¹⁰  Our group has recently demonstrated that intravenous MSC therapy enhances epithelial and endothelial repair after ventilation-induced ALI via a paracrine mechanism involving keratinocyte growth factor.¹¹ 

The optimal MSC delivery strategy, particularly the delivery route that provides the best balance between therapeutic benefit, invasiveness, and potential for harm, is not known. Intrapulmonary delivery may be more attractive than systemic administration for lung diseases, in that larger numbers of cells may be administered directly to the injury zone. Clinically, local cell delivery can be achieved by direct injection via the endotracheal tube in ALI/ARDS patients receiving ventilatory support. Furthermore, the type of lung injury, whether pulmonary or extrapulmonary, may be important, with endothelial injury perhaps treated best by intravenous MSC therapy whereas epithelial injury may be treated optimally via the intratracheal route.¹²  The safety of intravenous MSC infusion is clear from clinical trials in human disease, such as in myocardial infarction,¹³  graft versus host disease¹⁴  and stroke,¹⁵  and in preclinical models of ALI/ARDS.^9,10  Moreover, the ability of MSCs to home to injured tissues¹⁶  may obviate the need for local delivery strategies, and may result in localized therapeutic benefit after systemic delivery.¹⁷ 

We wished to determine the efficacy of intratracheal MSC therapy, gain insights into the mechanisms underlying these effects, and compare the efficacy of this approach with that of intravenous MSC therapy.¹¹  We hypothesized that intratracheal delivery of MSCs would (1) enhance functional recovery and lung repair after VILI; (2) that these effects would be mediated via a paracrine mechanism; and (3) that the efficacy of intratracheal delivery would be similar to intravenous MSC therapy.

These experiments were approved by the Animal Ethics Committee at the National University of Ireland, Galway and were performed under license from the Department of Health and Children, Ireland. Specific-pathogen–free adult male Sprague–Dawley rats (Charles River Laboratories, Kent, United Kingdom) weighing between 350 and 450 g were used in these studies. A full description of the methods is available in Supplemental Digital Content 1, http://links.lww.com/ALN/A911.

Bone marrow was aspirated from the tibiae of Sprague–Dawley rats, and plated into tissue culture flasks, as previously described.¹⁸  Adherent cells were grown until 80% confluent and then trypsinized and culture expanded to passage 4, whereupon they were used for experiments. MSCs were characterized according to the international guidelines (see figures, Supplemental Digital Content 2, http://links.lww.com/ALN/A912 and Supplemental Digital Content 3, http://links.lww.com/ALN/A913).¹⁹  Fibroblasts, isolated from the dermis of Sprague–Dawley rats as previously described, were used as control cells.²⁰ 

Allogeneic rat MSCs (4 × 10⁶) were cultured in serum-free media for 24 h. After replacement of the medium, the subsequent serum-free medium was used as the conditioned medium. Fifteen milliliters of this medium was centrifuged through a 3,000 kd filter (Amicon, Billerica, MA) to reduce volume to 300 µl.²⁰ 

We used our established model of repair from VILI.²⁰  Rats were anesthetized with intraperitoneal ketamine 80 mg·kg⁻¹ (Ketalar; Pfizer, Cork, Ireland) and xylazine 8 mg·kg⁻¹ (Xylapan; Vétoquinol, Dublin, Ireland). Tail vein intravenous access was obtained, and anesthesia was maintained with Saffan^® (Schering Plough, Welwyn Garden City, United Kingdom) and paralysis with cis-atracurium besylate 0.5 mg·kg⁻¹ (GlaxoSmithKline, Dublin, Ireland). Animals then received high stretch ventilation (P_insp 35 cm H₂O, zero positive end-inspiratory pressure; rate 18 min⁻¹). After induction of significant injury, as evidenced by a 50% decrement in static compliance, ventilation was discontinued and the animals allowed to recover²⁰  (fig. 1).

After recovery, animals were randomized to receive (1) no therapy, (2) intratracheal vehicle (phosphate-buffered saline, 300 µl), (3) intratracheal fibroblasts (4 × 10⁶), (4) intratracheal MSCs (4 × 10⁶), (5) intratracheal conditioned medium (300 µl), or (6) intravenous MSCs (4 × 10⁶). The timing of MSC delivery was 15–30 min after cessation of high stretch ventilation, i.e., approximately 2.5–3 h after the initiation of VILI.

Animals were reanesthetized 48 h after VILI induction as described in rodent ventilator-induced injury protocol. A tracheostomy was performed, and static lung compliance assessment and arterial blood gas analysis performed as previously described.^21,22  After 20 min, Fi_O2 was increased to 1.0 for 15 min, and arterial PO₂ measured. After heparin (400 U·kg⁻¹; CP Pharmaceuticals, Wrexham, United Kingdom) administration, animals were euthanized by exsanguination under anesthesia. Immediately postmortem, the heart–lung block was removed and bronchoalveolar lavage (BAL) carried out.^23,24  BAL differential cell counts were performed. BAL protein was measured using a Micro BCA^TM Protein assay kit (Pierce, Rockford, IL).²⁵  BAL tumor necrosis factor-α, interleukin-1β, interleukin-6, and interleukin-10 concentrations were determined using enzyme-linked immunosorbent assays (R&D Systems, Abingdon, United Kingdom).²⁶  Wet:dry lung weight ratios were measured using the right lower lung lobe.²⁷  The left lung was fixed using paraformaldehyde, and histologic lung injury assessed using quantitative stereology.^23,27  The investigator performing the physiologic assessment was not blinded to group allocation; however, the investigators were blinded for all other assessments and assays.

Cell tracking studies were performed in the intravenous and intratracheal MSC groups. MSCs were labeled with red fluorescent marker (PKH26; Sigma, St. Louis, MO; see Supplemental Digital Content 1, http://links.lww.com/ALN/A911 and were then administered intravenously or intratracheally after VILI. At 1, 4, and 24 h post-MSC administration, lungs from rats were removed and digested to generate single-cell suspensions for subsequent flow-cytometry (see Supplemental Digital Content 1, http://links.lww.com/ALN/A911). Flow cytometry was performed using FACScan and CellQuest-Pro software (Becton Dickinson, Franklin Lakes, NJ).

Sigmastat 3.1 (Systat Software, San Jose, CA) was used for all statistical analyses. Sample size was determined based on our previous studies.¹¹  Data distribution was assessed using Kolmogorov–Smirnov tests. Data were analyzed by one-way ANOVA, followed by Student–Newman–Keuls, or by Kruskalis–Wallis followed by Mann–Whitney U test with the Bonferroni correction for multiple comparisons, as appropriate. Residual plots were used to validate underlying model assumptions. A P value of less than 0.05 (two-tailed) was considered to be significant.

Forty animals were entered into the experimental protocol. All survived the injury and subsequent treatment allocation. Eight animals were each entered into the fibroblast, vehicle control, intratracheal MSC, and intratracheal conditioned medium groups; four animals were each entered into the intravenous MSC and no therapy groups. There were no baseline between-group differences in terms of preinjury variables, the duration of injurious ventilation, or the extent of the lung injury produced (table 1). All animals in each group survived the VILI recovery protocol. There were no differences in arterial pH, PCO₂, bicarbonate, lactate, or mean arterial pressure among the groups at the end of the recovery period (table 2).

Intratracheal MSC administration facilitated restoration of arterial oxygenation, reducing alveolar-arterial oxygen gradient (P < 0.001) (fig. 2A) and increasing arterial oxygenation (P < 0.001) (table 2) compared with vehicle. Further functional recovery in lung physiology in response to intratracheal MSC therapy was demonstrated by significant improvements (P < 0.001) in respiratory system static compliance in comparison with vehicle (fig. 2B). Intratracheal MSCs improved lung microvascular permeability, decreasing lung wet:dry weights (P < 0.001) (fig. 2C) and reducing BAL protein concentrations (P = 0.006) (fig. 2D).

Intratracheal MSCs decreased total inflammatory cell counts in BAL fluid (P < 0.001) (fig. 3A), and substantially attenuated (P < 0.001) lung neutrophil and macrophages accumulation (fig. 3, B and C). MSC therapy altered the proportions of inflammatory cells recruited to the ventilator-injured lung. Interestingly, intratracheal and intravenous MSCs increased the proportion of alveolar lymphocytes, whereas conditioned medium increased the proportion of alveolar macrophages, although this latter effect was modest (fig. 3, C–F). Intratracheal MSC therapy decreased alveolar concentrations of tumor necrosis factor-α (P < 0.001) (fig. 4A) and interleukin-6 (P < 0.001) (fig. 4B). In contrast, intratracheal MSC therapy did not alter BAL interleukin-10 concentrations (P = 0.873) (fig. 4C). Alveolar concentrations of keratinocyte growth factor were modestly increased by MSC therapy, being significantly (P = 0.018) increased only with intratracheal MSC therapy (fig. 4D).

Intratracheal MSCs reduced alveolar thickening, as evidenced by decreased fractional alveolar tissue volume (P < 0.001), and enhanced the restoration of airspace volume, as evidenced by enhanced fractional alveolar air-space volume (P < 0.001) (fig. 5, A and B). Representative histologic sections of lung demonstrate the enhanced repair of the injured lung in the MSC-treated animals (fig. 5, C and D).

The efficacy of intratracheal MSC therapy in enhancing lung repair after injury was also seen with MSC conditioned medium (fig. 2A–D). Intratracheal MSC conditioned medium also resulted in a similar pattern of reduction in lung inflammatory cells and altered cytokine profile in response to VILI (figs. 3 and 4). These findings suggest a paracrine mechanism of action for these cells. Importantly, nonstem/stromal cells, i.e., fibroblasts, did not have any therapeutic effect.

The magnitude of the therapeutic effect of intratracheal MSCs was similar to that seen with intravenous MSC therapy (figs. 2–4). However, animals that received intravenous MSC therapy demonstrated an increase in BAL interleukin-10 concentrations (P < 0.001) (fig. 4C). This was not seen in animals that received intratracheal MSCs or intratracheal conditioned medium. Higher alveolar concentrations of keratinocyte growth factor were demonstrated in the intratracheal MSC group alone (P = 0.018) (fig. 4D). In addition, a greater number of administered MSCs were retained in the lung after intratracheal than after intravenous delivery at 1, 4, and 24 h post-MSC administration (table, Supplemental Digital Content 4, http://links.lww.com/ALN/A914).

MSCs exhibit considerable therapeutic promise for patients with ALI/ARDS. MSC therapy attenuated endotoxin-induced ALI when given during the injury phase in mice.^8,9  Cells derived from the bone marrow, including MSCs, have improved survival and reduced injury in preclinical models of systemic polymicrobial sepsis.^28,29  Human MSCs attenuate the decrement in alveolar epithelial fluid clearance via secretion of keratinocyte growth factor in ex vivo perfused human lung after endotoxin injury.¹⁰  Recently, we have demonstrated that intravenous MSC therapy enhances epithelial and endothelial repair after ventilation-induced ALI by a keratinocyte growth factor-dependent paracrine mechanism.¹¹  Although studies suggest that both the intratracheal and systemic MSC delivery routes may be effective in attenuating ALI,^8,30  the optimal delivery method remains unclear, and there are no data comparing these routes in the setting of repair after injury. In these studies, we report that intratracheal administration of MSCs restored lung function after ventilation-induced injury, that they appear to act via a paracrine mechanism, and that they are as effective in restoring lung function as intravenous MSC therapy.

Intratracheal MSC therapy enhanced lung repair after VILI, as demonstrated by a reduced alveolar-arterial oxygen gradient, improvements in lung compliance and alveolar-capillary permeability. Intratracheal MSC therapy also modulated the inflammatory response to injury, decreasing alveolar white cell and neutrophil counts, and decreasing alveolar tumor necrosis factor-α and interleukin-6 concentrations. Intratracheal MSC therapy also facilitated restoration of lung structure after stretch injury.

In our studies, both MSC and conditioned medium therapy decreased overall alveolar inflammatory cell infiltration. In contrast, intravenous and intratracheal MSC therapy increased the percentage of lymphocytes in the alveolar fluid. MSCs have well-described effects on T- and B-lymphocytes, decreasing the proliferation and activation of these cells, while enhancing the production of T-regulatory cells, a T-cell population that plays a role in down-regulation of inflammation and tissue repair.³¹  Although the composition of the T-cell population was not examined here, MSC/T-cell interactions seem to require cell contact.³¹  Our finding that this increase in the proportion of alveolar lymphocytes was not seen with MSC conditioned medium seems to support this. The significance of these findings is unclear, given that both MSCs and conditioned medium augmented lung repair in these studies.

Intratracheal MSC therapy seems to enhance repair after VILI by a mechanism that is paracrine dependent. Conditioned medium from MSCs was as effective as intratracheal MSC therapy in repairing the injured lung. MSC-conditioned medium also resulted in a similar pattern of reduction in lung inflammatory cells and altered cytokine profile in response to VILI. The finding that intravenous and intratracheal MSC therapies were equally effective in restoring lung function, despite markedly different lung accumulation profiles, demonstrates that their precise disposition within the animal was of lesser importance, and supports a paracrine mechanism of action. These findings are supported by previous data demonstrating that MSCs act in large part via the secretion of paracrine mediators.^10,32  MSC administration modestly increased alveolar keratinocyte growth factor concentrations, although it was only significantly increased after intratracheal administration. Keratinocyte growth factor improves alveolar epithelial wound repair, is secreted in excess by MSCs,¹¹  and has been implicated in the mechanism by which MSCs enhance pulmonary epithelial wound repair.¹¹  Intriguingly, Islam et al.³³  recently demonstrated that MSCs release mitochondria-containing microvesicles, which can restore lung epithelial function after injury, providing another potential mechanism by which the MSC “secretome” may restore lung function. The finding that nonstem cells, i.e., fibroblasts, did not have any therapeutic effect, suggests that the reparative effects of MSCs are a function of the stem/stromal cell properties of MSCs.

In this study, intratracheal MSCs therapy was as effective as intravenous MSCs in restoring physiologic lung function and facilitating recovery of structural integrity after severe VILI. Intratracheal MSC therapy improved measures of alveolar epithelial and endothelial barrier function, including wet:dry ratios and BAL protein concentrations to a similar extent to that seen with intravenous MSC therapy. Both intravenous and intratracheal MSC therapy decreased alveolar concentrations of the key proinflammatory cytokines tumor necrosis factor-α and interleukin-6. Of interest, intravenous MSC therapy enhanced alveolar interleukin-10 concentrations, a finding not seen in animals that received intratracheal MSCs or MSC medium. This finding, which has been previously reported,²⁰  suggests that MSCs enhance interleukin-10 secretion via an interaction with one or more cell types encountered in the circulation. Intravenous MSCs have been demonstrated to enhance macrophage interleukin-10 secretion in the setting of systemic sepsis.²⁸  In our study, the increased alveolar interleukin-10 concentrations in animals that received intravenous MSCs are not explained by alterations in alveolar macrophage proportion or absolute numbers. The significance of MSC-induced interleukin-10 secretion in this repair model is unclear, given the fact that both intravenous and intratracheal MSCs were equally efficacious in repairing the injured lung after VILI.

This study suggests that the intratracheal route is a viable alternative to the intravenous route for MSC delivery to promote repair in the lung. These results extend previous findings demonstrating that intratracheal MSC therapy is effective in attenuating the injury phase of ALI.^8–10  The potential advantages of the intrapulmonary route of delivery include the ability to deliver larger numbers of cells directly to the injury zone, their ease of administration in the clinical setting via the tracheal tube, and the potential for reduced systemic effects. In contrast, intravenous MSC delivery is not without its risks. Although intravenous MSCs can home to injured organs³⁴  including the lung,³⁵  these MSCs are also trapped in the vasculature of the lung,³⁴  potentially leading to pulmonary capillary plugging, reduced pulmonary vascular compliance, pulmonary hypertension, and right ventricular failure. Intracoronary MSC administration after myocardial infarction caused microvascular plugging, which reduced coronary blood flow, underlining the importance of these concerns.³⁶  The development of pulmonary hypertension may be particularly deleterious in patients with ALI/ARDS.³⁷  Consequently, intratracheal MSC delivery may be a useful therapeutic approach in ALI/ARDS patients.

A number of limitations deserve consideration. First, our studies were conducted in a preclinical rodent model and caution is required in considering clinical relevance. Second, baseline data are not available on these animals. However, we have characterized the effect of high lung stretch in detail in this model in a previous publication.²⁰  Last, we did not examine the effects of MSCs in protectively ventilated or unventilated animals, as the effects on uninjured animals would be expected to be limited.

In conclusion, intratracheal MSCs enhance lung repair after VILI to a similar extent to that seen with intravenous administered MSCs. The mechanism of action of the intratracheal MSCs seems to be due to MSC secretion of paracrine factors. Intratracheal MSC therapy seems to have considerable promise for the treatment of patients suffering from VILI and ARDS.

The authors acknowledge Georgina Shaw, B.Sc., Research Technician, Regenerative Medicine Institute, National University of Ireland, Galway, Ireland, for her assistance in harvesting and preparing the mesenchymal stromal cells used in these studies.