Intrapulmonary autologous transplant of bone marrow-derived mesenchymal stromal cells improves lipopolysaccharide-induced acute respiratory distress syndrome in rabbit

Lung disorders have significant morbidity and mortality rates worldwide, both in humans and in animals. Acute respiratory distress syndrome (ARDS) is one of the leading causes of respiratory failure around the world. Although early diagnosis, timely medical care, and treatment may lead to improvement of symptoms, the signs return after a period of time. Causes of ARDS are different. It can be caused by direct or indirect damage to the lung epithelium. ARDS is described by severe hypoxemia, decreased pulmonary compliance, diffuse alveolar damage, and bilateral pulmonary infiltrates after cardiac edema [1, 2] and confirmed by a combination of clinical, physiological, and chest imaging parameters. Pulmonary inflammation with disruption of the mechanism of the alveolar–capillary barrier is an important direct cause of ARDS [3, 4]. Therapeutic approaches include mechanical ventilation, neuromuscular blocking agents, fluid management, drug and antimicrobial therapy, and prone positioning [3, 5, 6]. These therapeutic strategies have a supportive role and cannot prevent the progression of the disease [7‐10]. The ultimate approach is lung transplant, but it has many problems for recipient patients such as lack of suitable donors and the use of immunosuppressive drugs over a lifetime to prevent rejection of the transplant [11]. Therefore, the recognition of new therapeutic approaches such as stromal cell therapy is essential [12]. MSCs confer immunomodulatory and anti-inflammatory effects, enhance bacterial clearance, reduce cell injury and death, and are angiogenic [5, 13]. The mechanism of the MSCs’ effects includes several pathways mediated through differentiation, proliferation, soluble intermediate release, extracellular vesicles, transfer of organelles, and direct cell-to-cell contact, which decrease activation of inflammatory cell secretion of paracrine mediators [14, 15]. Recent studies have shown positive effects of MSC-based therapy for ARDS. Induction of inflammation by the LPS of Escherichia coli O55:B5 is one of the best and simplest methods for making an experimental model of ARDS. Although the ARDS animal models cannot reflect human ARDS accurately, the rabbit model is similar and hence suitable for translating the results from pilot to clinical conditions [16]. Anatomical, physiological, genetic, and biochemical similarity to humans simulates human lung disease, and as the rabbit is easy to handle, it is considered as a suitable model for pulmonary research [17, 18]. Moreover, the rabbit serves as an excellent platform for treatment based on stromal cells [19, 20]. Thus, in this study, the rabbit was used as a model for causing ARDS, and then it was treated with stromal cells. The aim of this study was evaluation of therapeutic potential intrapulmonary administration of BM-MSCs in an experimental model of E. coli LPS-induced ARDS in the rabbit.

BM-MSCs are an ideal choice for cell therapy because there are fewer complications for cell isolation and also BM autologous cells that are capable of eliminating immune response and transplant rejection [22]. MSCs have positive effects in the repair of ARDS [23].

Although fibroblasts play a role in normal and pathological repair, and an accumulation of fibrocytes, fibroblasts, and myofibroblasts in the alveolar compartment, leading to excessive deposition components of the extracellular matrix but also according to prior findings the effect of them remains controversial [23‐25] and unknown, so that reduction of its deposition or enhancement of its degradation could be treatment strategies. [24]

The results of this study showed that the BM-MSCs significantly decreased the severity of clinical symptoms induced by LPS, the number of inflammatory cells in blood and BAL, and balanced the values of arterial blood gases and cytokines. On the CT scans, a significant decrease in the Hounsfield units was observed, which is indicative of an increase in aerated volume of the lung in the treatment group. The echocardiographic parameters did not reveal a significant difference in the treatment group, and compared with the two groups together it was significant. Also, the histopathology demonstrated reduction in the infiltration of inflammatory cells and pulmonary hemorrhage and edema in the recipients of BM-MSCs.

In natural conditions, cells from bone marrow migrated to chemotactic gradients, but the amount of engraftment was low, and use of exogenous stromal cells can be helpful to this mechanism. The exact mechanisms of MSCs’ actions are not precise [25], and sometimes the results of stromal cell research using animal models are incompatible with each other [23]. However, three mechanisms have been defined for MSCs’ actions consisting of differentiation, cell–cell contact and paracrine function via the soluble factors [25, 26]. Researchers are likely to focus on manipulation of inflammatory pathways and optimizing lung repair while preventing ARDS progression [6]; whereas management of inflammatory pathways forbids the development of ARDS, some researchers believe in the immunomodulatory effects of MSCs [6]. Most trials have used local delivery of autologous BM-MSCs with the aim increasing the concentration of growth factors and cytokines in damaged tissue to improve possible engraftment and repair [23]. For the first time, Gupta et al. reported effects of the local delivery of MSCs in ARDS that are consistent with this study [26].

The anti-inflammatory role of MSCs may vary in humans and animals, but the useful effects of MSCs have been shown in several animal models [27] and indicate the significant role of identification feature of MSCs [22]. LPS of gram-negative bacterial wall binds to the CD14/TLR4/MD2 receptor complex, activating pathway and transcription of some inflammation- and apoptosis-related genes and activating innate immune response [18, 25] that produces the acute phase of ARDS. Therefore, the LPS-induced animal models could be suitable for cell therapy. In prior findings, only a few animal models have been used to investigate the mechanism of MSC therapy in ARDS, most of which used rat and mouse [25, 28‐31]. Although animal models cannot show all complications of human ARDS, a rabbit can display important physiological and pathological features of human ARDS and can be useful for novel therapeutic strategies [18]. As previously described in this study, we used LPS from E. coli strain O55:B5to induce an experimental model of ARDS in the rabbit.

Our results displayed inflammatory cells downregulated by BM-MSCs, which can improve the function of the alveolar-capillary membrane, in agreement with the results of Xiang et al. on improving pulmonary microvascular permeability with MSCs [32]. The reduction of heterophils, macrophages, and the number of total cells were observed in the treated group, unlike the results of other research [26]. The complete blood count findings indicated that despite intrapulmonary administration of LPS and MSCs, there is a relationship between the numbers of intravascular and intra-alveolar heterophils and lymphocytes. Direct administration of LPS effects on gas exchange process and induces acute hypoxemia [33]. The results of studies suggest that MSC transplant can improve hypoxemia via reducing alveolar-arterial oxygen gradient [29, 30] which is in agreement with the present study. But the research results of Moodley et al., (2016) conflicted with this study [27]. The difference in injury model and type of cell therapy may be the cause of the variable results. There are not any studies that have reported clinical signs, including HR, RR, and RT, after stromal cell therapy; this study is the first report in which clinical signs were evaluated using a previously described scoring system.

The protective effects of BM-MSCs on ARDS are a result of the immune regulation function and inhibitory T-cell function [31]. Monocytes and macrophages can release inflammatory factors such as TNF-α that play a role in phagocytes of the necrotic and apoptotic cells [34]. Because MSC soluble factors may be the therapeutic basis of MSCs, this result is confirmed by recent reports that demonstrated immunomodulatory properties of MSCs [26]. MSCs’ effects are explained by a shift from a proinflammatory to an anti-inflammatory response [26]. Reduced cytokines of proinflammatory (TNF-α and IL-6) and increased cytokines of anti-inflammatory (IL-10) in BAL and plasma samples play principal roles in the treatment mechanism of ARDS [26, 29, 30]. In the present study, the TNF-α concentration decreased following MSC therapy, which is similar to many studies [31]. The IL-10 level increased after the use of MSCs and inhibited antigen-presenting cell function and inflammation and enhanced tolerance, but our findings are not similar to some other studies [26, 30]. Beneficial effects of MSCs may be mediated by a decrease of TNF that indicated the attenuation of the inflammation [26]. Prostaglandin E2 (PGE2) has a critical role in IL-10 and IL-6 secretion from macrophages [31]. It is possible that TNF- and IL-6-dependent PGE2 production plays a major role as a clinical sign and in particular hyperthermia in the acute phase of inflammation [34]. TNF has roles in local inflammation complications such as leukocyte stimulation (neutrophils and macrophage) in BAL and lung endothelium that was shown in the pathology. Also, TNF has roles in systemic complications such as hyperthermia, C-reactive protein, leukocytosis, necrosis, and apoptosis, decreasing appetite, and even decreases of cardiac output and vascular permeability and increases in edema and hypotension.

In this study, CT scans and echocardiography were used to follow improvement and management of ARDS during BM-MSC therapy. One particular feature of ARDS is lack of aerated lung volume caused by inflammation and edema, and total lung volume reduction (air + parenchyma) mostly in the lower lobes can explain hypoxemia, low respiratory compliance, and alveolar dead space and increased pulmonary vascular permeability [35, 36]. CT scanning is a common clinical diagnostic tool, a specific, repeatable, and noninvasive technique for diagnosis that until now has rarely been used as a research tool, and there is not any valid data of the distribution region of the existence or absence of air in alveolus during ARDS and especially after cell-based therapy. Our findings based on CT scanning are matched with the diagnosis of pathology and all the other outcomes. Therefore, it can be said that CT scanning is a suitable method for evaluation of the stromal cell therapy effects in pulmonary inflammation and edema, but further studies are needed in this field. Interactions between lung, right ventricle, and pulmonary circulation are critical in ARDS. Serial echocardiographic measurements have a potential clinical diagnosis in the early stage ARDS as a prognostic and therapeutic method. We showed that ARDS causes changes in echocardiographic parameters and reduces cardiac function, whereas transplant of BM-MSCs was able to prevent these changes.

We demonstrated that MSCs could attenuate the inflammation by reducing pathological lung changes [31], but in some research, no significant data were reported regarding pathology. This variation may be caused by short study duration so that further studies are needed. Our histopathological examination, like other findings from imaging and all of the laboratory results, showed that BM-MSC transplant could improve ARDS.

Many studies on cell-based therapies in ARDS have been done, but most of them focused on molecular and signal tests. Although these studies could clear pathways, they are still far from tissue function. In this study, we tried to explain the MSCs’ effects on organ function. We investigated effects of BM-MSCs in an experimental model of ARDS and confirmed that MSCs decrease inflammation and improve alveolar fluid clearance and have a protective role in ARDS. Improvement in clinical signs, the decrease of inflammatory cells in blood and BAL, the balance in blood gases and cytokines, the decrease in the Hounsfield units, no changes in echocardiographic parameters. and the reduction of pulmonary hemorrhage and edema in pathology were observed. Despite these results, subsequent studies are required to confirm the decrease in inflammation, and physiological parameters over the long term and many experiments should be performed until stromal cell therapy is validated as a method of routine clinical treatment.