Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), are critical illnesses caused by excessive and uncontrolled systemic inflammatory responses to direct or indirect lung injury. ALI is defined as the acute onset of diffuse bilateral pulmonary infiltrates by chest radiograph, and with a PaO2/FiO2 ≤ 300mmHg or without clinical evidence of left atrial hypertension (1). Those with more severe hypoxemia (PaO2/FiO2 ≤ 200mmHg) are considered to have ARDS. While according to the Berlin definition, ARDS is divided into three categories in terms of the degree of hypoxemia: mild (PaO₂/FiO₂ = 200–300 mmHg); moderate (PaO₂/FiO₂ = 100–200 mmHg); and severe (PaO₂/FiO₂ < 100 mmHg) (2). ALI and ARDS are characterized by severe intractable hypoxemia, hypoxic respiratory failure, alveolar and interstitial pulmonary edema, decreased pulmonary compliance, and increased pulmonary vascular permeability (3, 4). The main pathological changes associated with ALI/ARDS include damage to alveolar epithelial cells and alveolar capillary endothelial cell barriers, the exudation of protein-rich edema fluid from the alveolar lumen, and the infiltration of inflammatory cells such as neutrophils and macrophages (5).

ALI/ARDS may be caused by a variety of factors including shock, severe sepsis, pulmonary contusion, gastroesophageal reflux, aspiration of gastric contents, pneumonia, drug toxicity, blood transfusion, acute pancreatitis, ischemia-reperfusion, and drowning (6).

The pathogenesis of ALI/ARDS is complex, and the main pathogenic mechanisms are excessive inflammatory response caused by the massive release of pro-inflammatory cytokines and damage to alveolar epithelial cells resulting from the excessive activation of multiple immune cells. The lung tissue can be stimulated by multiple factors to trigger infiltration of various inflammatory cells such as macrophages and neutrophils, thereby releasing large amounts of pro-inflammatory cytokines and inflammatory mediators. This disrupts the integrity of alveolar epithelial cells and the pulmonary capillary endothelial cell barrier, leading to pulmonary edema and alveolar hemorrhage (6, 7). ARDS can be classified into two subtypes, hyperinflammatory and hypoinflammatory, based on clinical features such as oxygenation index and inflammation-related biomarkers such as the levels of IL-6, IL-8, IL-18, TNF-α, and ACE2. The hyperinflammatory subtype of ARDS has poor prognosis and high mortality (8).

Treatment of ALI/ARDS is supportive. The current clinical treatment of ALI/ARDS is based on lung-protective mechanical ventilation and fluid management therapy (9), supplemented by glucocorticoids (10, 11), surfactants (12, 13), N-acetylcysteine (14, 15), statins (16, 17), β2 agonists (18), neuromuscular blockade (3, 19), extracorporeal membrane pulmonary oxygenation (20) and prophylaxis for venous thromboembolism (21). In addition, for infections such as sepsis-induced ARDS, antimicrobial drugs can be used pertinently (22). Moreover, for ARDS caused by blood transfusion, the dose-response relationship between the amount of blood products transfused in patients and the risk of developing ARDS suggested that restrictive transfusion policies may reduce the incidence of ARDS in these patients (23, 24). Although these supportive treatments can improve patients’ symptoms to a certain extent, they do not significantly improve the prognosis, and the associated mortality remains high. An international multicenter prospective cohort study indicated that the intensive care unit (ICU) admission of ARDS patients was 10.4%, with an overall mortality rate of 35%–46% (25). Another large clinical trial found a 43% mortality rate at 90 days in patients with ARDS (26). Even patients in recovery may face long-term cognitive impairment and impaired quality of life (27). Moreover, although mechanical ventilation is the basic and important methods for the treatment of ALI/ARDS, long-term/excessive mechanical ventilation may lead to ventilator-related lung injury (VILI) and even pulmonary fibrosis (28–33). Therefore, there is an urgent need to explore more effective and safe therapeutic measures for ALI/ARDS.

MSCs are pluripotent, self-renewing stem cells with multi-directional differentiation potential and migration ability. MSCs originate from a variety of organs and tissues such as bone marrow, adipose, muscle, umbilical cord, and placenta tissues. Based on these characteristics, MSCs are widely used in the field of tissue regeneration (34, 35). MSCs can regulate immune homeostasis, reduce lung inflammation, repair tissue damage, and promote tissue regeneration, making them promising for the treatment of ALI/ARDS (36–39).

The presence of macrophages in lung tissue plays a critical role in the inflammatory response to ALI/ARDS. Macrophages in lung tissue mainly include two subpopulations: alveolar macrophages and interstitial macrophages. Alveolar macrophages, which are more abundant than interstitial macrophages, are the first line of defense against foreign invading factors and play an important role in host defense and the maintenance of immune homeostasis in the local microenvironment of lung tissue (40–42). In response to stimulation by foreign pathogens, alveolar macrophages can release a variety of inflammatory factors and chemokines to initiate a cascade of amplified inflammatory responses within lung tissue and mediate lung tissue injury.

Macrophages have good plasticity and can polarize into different phenotypes under different environmental conditions, including classically activated M1 macrophages and alternatively activated M2 macrophages ( Figure 1 ). M1 macrophages produce a large amount of pro-inflammatory cytokines, which cause tissue damage while facilitating the host immune clearance of pathogens. M2 macrophages mainly secrete anti-inflammatory cytokines, which facilitate wound healing and the repair of tissue damage (43–45).

The M1 and M2 phenotypes of macrophages can be interconverted under certain conditions. M1 and M2 macrophages can effectively avoid excessive inflammatory responses that cause tissue damage by maintaining the immune homeostasis of the lung microenvironment. In the progression of ALI/ARDS, the balance of M1 and M2 macrophages can effectively remove harmful substances and over-produced pro-inflammatory cytokines from the body to promote the repair of lung tissue damage. However, the loss of balance may exacerbate lung injury and worsen ALI/ARDS. MSCs can modulate macrophage function by regulating the polarization of macrophages; thus, MSCs show promise for the treatment of ALI/ARDS and the improvement of ALI/ARDS patient prognosis (46–50).

Macrophages are usually polarized to the M1 phenotype in response to microbial stimuli including lipopolysaccharide (LPS) and Th1-related cytokines such as IFN-γ and TNF-α (44) ( Figure 1 ). M1 macrophages highly express CD86, CD80, and other surface markers. M1 macrophages also secrete numerous inflammatory mediators and cytokines such as TNF-α, IL-1, IL-6, IL-12, iNOS, reactive oxygen species (ROS), monocyte chemotactic protein 1 (MCP-1), and macrophage inflammatory protein 2 (MIP-2). These compounds lead to a strong inflammatory response and Th1 immune response and cause tissue damage while participating in pathogen clearance (51). In the early stage of ALI/ARDS, alveolar macrophages are M1-polarized and release various pro-inflammatory factors and harmful mediators while clearing pathogenic microorganisms and recruiting neutrophils and other inflammatory cells. Eventually, the excessive accumulation of pro-inflammatory cytokines and inflammatory cells lead to lung tissue injury (52).

M2 macrophages are usually induced by IL-4, IL-13, TGF-β, and macrophage colony-stimulating factor (M-CSF; Figure 1 ). Activated M2 macrophages highly express arginase 1 (Arg-1) and specific surface markers such as CD206, CD163, chitinase 3-like 3 (Ym-1) and found in inflammatory zone 1 (Fizz1). M2 macrophages also secrete large amounts of the anti-inflammatory cytokine IL-10, which inhibits inflammatory cell aggregation and negatively regulates the production of anti-inflammatory factors (53).

M2 macrophages can be further divided into four subtypes: M2a, M2b, M2c, and M2d ( Figure 1 ). The activation of M2a macrophages is induced by IL-4 and IL-13, which can be involved in allergic reactions, wrapping and killing parasites. M2b macrophages are induced by immune complexes (ICs), Toll-like receptor (TLR) ligands, and IL-1R agonists. M2b macrophages, which secret high levels of IL-10 and low levels of IL-12, which is conductive to the Th2 immune responses. Glucocorticoids, IL-10, and TGF-β are involved in the induction of M2c macrophages. Activated M2c macrophages negatively regulate the production of anti-inflammatory cytokines involved in tissue remodeling and matrix deposition (43, 44, 54–56). M2d macrophages are synergistically induced by TLRs and adenosine A2a receptor agonists or by IL-6. M2d macrophages have the characteristics of tumor-like macrophages and can participate in tumor-related immune regulation, tumor growth, and tumor angiogenesis (57–59). In addition, some investigators also described a new subpopulation of pro-resolving macrophages, namely CD11b^low macrophages that emerge during the resolution of zymosan-induced peritonitis in mice. These macrophages secrete pro-resolving mediators and are generated by M2-like macrophages in vivo and in vitro after phagocytosis of apoptotic leukocytes (60). Another study suggested that pro-resolving macrophages could produce antiangiogenic mediators such as endostatin, which could halt angiogenesis and restore tissue structure (61). In summary, macrophages are highly plastic cells. They may exert a vast “spectrum” of functions characterized by an array of different macrophage phenotypes/subtypes (62).

M2 macrophages are key regulators of tissue repair during ALI/ARDS recovery (63, 64). M2 macrophages can release anti-inflammatory cytokines, inhibit the production of pro-inflammatory mediators, and remove apoptotic neutrophils from inflammatory sites to promote lung injury repair. M2 macrophages can also suppress the expression of iNOS and prevent the production of ROS, thereby reducing damage to alveolar epithelial cells and promoting the recovery of host tissues (52, 65–67).

In summary, M2 macrophages can suppress inflammation, promote tissue remodeling, and participate in angiogenesis and immune regulation. M2 macrophages are also thought to promote fibrosis. The TGF-β1 secreted by M2 macrophages can trigger fibroblast activation and extracellular matrix-producing myofibroblast development while promoting the resolution of inflammatory response to drive fibrosis (68, 69). Maintaining the appropriate balance of M1 and M2 macrophages in vivo thus provides a target for the treatment of ALI/ARDS.

MSCs can home to injured sites to reduce inflammatory response. MSCs differentiate toward type II alveolar epithelial cells, which participate in the tissue repair process (74). However, some studies suggested MSCs retained a low number and had a short duration in the injured tissue after transplantation (75–79). Therefore, MSCs homing and differentiation are not the key mechanisms of their effects in ALI/ARDS. Rather, paracrine action may be the primary mechanism of MSCs involvement in tissue injury and repair ( Figure 2 ).

MSCs secret soluble factors such as chemokines, cytokines, and growth factors, which exert paracrine effects to promote tissue repair and regeneration (80, 81). The paracrine action of MSCs plays a crucial anti-inflammatory and immunosuppressive role in immune cells. Wakayama et al. reported that MSCs promoted macrophage polarization to the anti-inflammatory phenotype via a paracrine mechanism, expressed high levels of CD206 and Arg-1, attenuated pro-inflammatory responses, and reduced bleomyclin-induced ALI in mice (82).

MSCs from dental capsules promoted anti-inflammatory polarization via the paracrine secretion of TGF-β3 and thrombospondin-1 to upregulate the level of anti-inflammatory factors, thereby reducing the pulmonary inflammatory response (72). Kwon et al. suggested that UC-MSCs could regulate the anti-inflammatory response by secreting decorin, a key regulator that polarizes inflammatory macrophages into anti-inflammatory macrophages (83). Kim et al. reported that the paracrine secretion of Pentraxin 3 from UC-MSCs enhanced the levels of anti-inflammatory macrophage markers and anti-inflammatory cytokines to improve hyperoxic lung injury (84). MSCs also attenuated lung inflammation by promoting the anti-inflammatory macrophage phenotype via the paracrine secretion of insulin-like growth factor (85). Prostaglandin (PGE2) secreted by MSCs induced an immunosuppressive anti-inflammatory phenotype in alveolar macrophages, increased the ability to produce IL-10, and inhibited the production of TNF-α (86). Other soluble factors such as Galectin-9 (87), tumor necrosis factor-stimulated gene 6 (TSG-6) (88), indoleamine 2,3-dioxygenase (89), and IL-6 (90) may also be involved in the regulation of macrophage polarization to combat inflammation.

MSC-derived extracellular vesicles can be divided into three main subtypes based on their biological properties and diameters: exosomes (30–150 nm), microvesicles (100–1000 nm), and apoptotic vesicles (50–5000 nm) (91–94) ( Figure 3 ). Exosomes are produced through the intracellular body pathway as follows. The cell membrane is invaginated to form early endosomes, which interact with vesicles formed by Golgi apparatus budding to form late endosomes. The late endosomes further develop into multivesicular bodies (MVBs) that contain intracellular vesicles. The MVBs fuse with the lysosomal membrane or cell membrane and degrade, releasing the contents (i.e., exosomes) into the extracellular environment through exocytosis (95, 96). The process of exosome formation is shown in Figure 3 . The endosomal sorting complex required for transport plays a key role in exosome biogenesis (97). The density of exosomes is approximately 1.13–1.19 g/ml, and exosomes are widely found in plasma, serum, urine, BALF, breast milk, saliva, tears, ascites, and other body fluids (98–104). Exosomes express tetraspanins (CD9, CD81, CD63, and CD82), TSG10, heat-shock proteins, and apoptosis-linked gene 2-interacting protein X (Alix) (105–107). Exosomes carry proteins, lipids, and RNAs that mediate intercellular communication between different cell types, participate in the regulation of cellular activity, and perform a variety of biological functions ( Figure 3 ) (108–111). Moreover, exosomes derived from MSCs can act as carriers of transported miRNA, enabling communication with recipient cells; thus, MSC-derived exosomes can affect disease pathogenesis along with tissue repair and regeneration (112, 113).

MSC-derived exosomes can attenuate ALI/ARDS by regulating macrophage polarization. Tian et al. found that adipose MSC-derived exosomes inhibited TLR4 expression to promote macrophage polarization and attenuate septic lung injury in mice (114). Song et al. reported that miR-146a expression was upregulated in MSC-derived exosomes after IL-1β stimulation, leading to macrophage polarization into the anti-inflammatory phenotype and increased survival in mice with sepsis-induced lung injury (115). Bao et al. demonstrated that MSC-derived exosomes can regulate alveolar macrophage pro-inflammatory polarization and block the pro-inflammatory pathway of macrophages, thereby reducing radiation-induced lung injury in mice (116). Lastly, Li et al. reported that the intratracheal administration of MSC-derived exosomes decreased pro-inflammatory polarization in alveolar macrophages while increasing anti-inflammatory polarization, leading to reduced pulmonary edema and pulmonary dysfunction along with decreased secretion of inflammatory factors such as IL-1β, IL-6, and TNF-α (117).

Macrophage polarization is closely related to metabolic status. Pro-inflammatory macrophage polarization is characterized by enhanced glycolysis and pentose phosphate pathways and decreased oxidative phosphorylation. In contrast, anti-inflammatory macrophage polarization is characterized by enhanced oxidative phosphorylation, and anti-inflammatory macrophages rely on oxidative phosphorylation for energy production (118–121). MSCs can induce anti-inflammatory macrophage polarization and promote tissue repair through metabolic reprogramming (122). Deng et al. found that BMSC-derived exosomes inhibited pro-inflammatory polarization and promoted anti-inflammatory polarization in mice alveolar macrophages (63). The authors further demonstrated that BMSC-derived exosomes reduced LPS-induced lung pathological injury by inhibiting the expression of hypoxia-inducible factor 1-α and downregulating the expressions of key proteins of macrophage glycolysis, thereby regulating macrophage polarization and inflammatory response.

In addition to increased lung epithelial and endothelial barrier permeability, the inflammatory response of ALI/ARDS can also cause mitochondrial dysfunction in lung tissue (123). MSCs may provide mitochondria to damaged cells, thereby enhancing cellular bioenergetics and improving organ dysfunction in ALI/ARDS and other inflammatory diseases. MSCs have been shown to restore mitochondrial function and alveolar bioenergetics through functional mitochondrial transfer, promote pulmonary epithelial injury repair, and improve alveolar capillary barrier permeability (124, 125).

Jackson et al. found that the extensive mitochondrial transfer of MSCs to macrophages enhanced macrophage phagocytosis and played an antibacterial role in a model of lung injury caused by E. coli-induced pneumonia (126). The mitochondrial transfer of MSCs plays a key role in regulating macrophage phenotype; Morrison et al. found that MSCs promoted macrophage oxidative phosphorylation through extracellular vesicle-mediated mitochondrial transfer and increased the expression of the anti-inflammatory macrophage marker CD206, thereby converting macrophages into the anti-inflammatory phenotype and ameliorating lung injury (73). Moreover, MSCs-derived exosomes can donate mitochondria components, improve macrophage mitochondrial integrity and oxidative phosphorylation levels, transform macrophages into an anti-inflammatory phenotype, and restore the metabolic and immune homeostasis of alveolar macrophages and reduce lung inflammation (127).

MSCs undergo necessary apoptosis and releases apoptotic vesicles during therapeutic application, apoptotic MSCs have unique anti-inflammatory properties and immunomodulatory effects (128). Apoptotic umbilical cord MSCs were able to reduce inflammatory exudates and vascular permeability in ALI rat lungs more effectively than normal umbilical MSCs. It could also more effectively reduce the level of pro-inflammatory factors and could more effectively increase the expression of anti-inflammatory cytokine IL-4, which was able to reduce the degree of pathological damage in rats with ALI (129). In addition, apoptotic MSCs could also exert a beneficial role in sepsis and allergic airway inflammation (130, 131).

Macrophages and monocytes that phagocytose apoptotic cells are capable of producing anti-inflammatory mediators (132, 133). When graft-versus-host disease patients were infused with in vitro-generated apoptotic MSCs, it was observed that recipient phagocytes engulfed the apoptotic MSCs and produced indoleamine 2,3-dioxygenase, thereby achieving an immunosuppressive effect (134).

In an animal model of ovalibumin-induced allergic asthma, the investigators showed that apoptotic MSCs exerted immunosuppressive effects in the lungs and inhibited allergic asthma to a similar extent as administration with viable MSCs (135). Besides, apoptotic MSCs inhibited the inward flow of eosinophils in BALF and the production of specific IL-5 and IL-13 in ovalbumin-induced allergic asthma to a similar extent as surviving MSCs, which indicated that the immunosuppressive effects of MSCs in the lungs did not require MSCs to retain survival (135). Moreover, in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalitis models, BKX-MSCs (depletion of the apoptotic effectors BAK and BAX) could not reduce circulating inflammatory monocytes that infiltrated the central nervous system to cause tissue injury, compared to untreated MSCs, which suggested that apoptotic MSCs were necessary for efficient in vivo immunosuppression and therapeutic effect (135).

The apoptotic and efferocytosis effects of MSCs could regulate macrophage phenotype polarization. For example, a study showed that efferocytosis of adipose-derived MSCs could alter the macrophages phenotype toward regulatory and anti-inflammatory phenotype (136). Another study suggested that MSC‐derived apoptotic vesicles exerted extensive regulatory effects on macrophages at the transcription level, which contributed to macrophage polarization towards the anti‐inflammation phenotype in type 2 diabetes treatment (137). Moreover, the apoptosis and efferocytosis of MSCs could induce the alterations of metabolic and inflammatory pathway in alveolar macrophages, thus affecting immunosuppression and reducing disease severity (135). Therefore, the apoptotic and efferocytosis effects of MSCs may play a beneficial role in the treatment of ALI/ARDS by modulating the polarization of macrophage phenotype, and more preclinical and clinical studies are still needed to confirm this idea in the future.

In summary, MSCs may regulate macrophage polarization through mechanisms such as paracrine soluble factors, exosomes, modulation of metabolic reprogramming and mitochondrial transfer, apoptotic and efferocytosis effects ( Table 1 ). The regulation of macrophage polarization by MSCs may be a potential and promising therapy for the treatment of ALI/ARDS.

The NF-kB pathway is widely recognized as a pro-inflammatory signaling pathway that participates in host immune response. The activation of inflammatory stimuli such as LPSs leads to the release and nuclear translocation of NF-kB (138, 139). The NF-kB signaling pathway plays an important role in the regulation of macrophage polarization (140, 141).

MSCs can regulate macrophage polarization through NF-kB signaling. Gao et al. demonstrated that co-culturing LPS-induced mice macrophages with MSCs decreased TNF-α expression and enhanced IL-10 and Arg-1 expression; the authors also found that the activity of the NF-kB pathway was inhibited in MSC-treated mice macrophages (142). Wang et al. reported that MSC-derived extracellular vesicles regulated macrophage polarization, attenuated lung injury, and improved pulmonary function by inhibiting the activation of the NF-kB pathway (143). MSCs also attenuated burn-induced ALI by inhibiting the activation of the TLR4/NF-kB signaling pathway via paracrine TSG-6 and transforming macrophages from the pro-inflammatory phenotype to the anti-inflammatory phenotype (144). In a multidrug-resistant Pseudomonas aeruginosa-induced ALI mice model, MSC-derived extracellular vesicles modulated the balance between pro-inflammatory and anti-inflammatory macrophages by inhibiting the activity of the NF-kB signaling pathway, leading to the downregulation of the pro-inflammatory macrophage markers iNOS and IL-12 and the upregulation of the anti-inflammatory macrophage markers Arg-1 and IL-10; these effects reduced the bacterial load and inflammatory response, resulting in decreased mice mortality (145).

Notch signaling is a conserved and important signal transduction pathway that mediates intercellular communication and plays a role in many cell types and different stages of development (146). The Notch signaling pathway is closely associated with inflammation and immune function (147). The Notch signaling pathway also plays a key role in the activation of macrophage polarization. The activation of the Notch signaling pathway promotes pro-inflammatory macrophage polarization and increases the expressions of pro-inflammatory macrophage markers (148–150). Bai et al. found that adipose MSC-derived extracellular vesicles regulated LPS-induced pro-inflammatory polarization by inhibiting the Notch signaling pathway and attenuating sepsis-induced lung injury and inflammatory cell infiltration in lung tissue (151).

Nrf2 is a transcription factor that regulates oxidation and inflammation to prevent oxidative damage and modulate apoptosis. Several anti-inflammatory factors can promote the nuclear translocation of Nrf2 and increase the expression of downstream HO-1 protein (152). The activation of the Nrf2/Ho-1 signaling pathway inhibits oxidative stress and inflammatory responses in sepsis-induced organ injury (153). The Nrf2/HO-1 signaling pathway plays a crucial role in regulating macrophage polarization (141, 154, 155). MSCs exert anti-inflammatory effects by activating the Nrf2/HO-1 signaling pathway, promoting anti-inflammatory macrophage polarization, inhibiting oxidative stress, and facilitating lung injury repair, which may be related to the expression of stanniocalcin-2, which has antioxidant properties, in MSCs (71).

The Janus family of kinases (JAK) is composed of four main members, JAK1, JAK2, JAK3 and Tyk2, which is non-receptor protein tyrosine kinase by nature (156, 157). JAK family kinases can phosphorylate signal transducers and activators of transcription (STATs) to regulate the expression of related genes; this pathway is called the JAK-STAT signaling pathway, The JAK-STAT signaling pathway can modulate cell proliferation, differentiation, apoptosis, and immune function (158). The STAT family includes seven members (STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6) that play crucial roles in many cellular functions (159, 160). STAT1 is the most important mediator of the IFN-γ-induced polarization of pro-inflammatory macrophages (44, 161, 162). The inhibition of JAK/STAT1 signaling can attenuate the activation of pro-inflammatory macrophages (163–165). STAT3 is an important transcription factor that activates macrophages and increases inflammation (166–168). STAT6 is a major transcription factor for IL-4- or IL-13-mediated signaling and plays an important role in anti-inflammatory macrophage polarization (169). MSCs can modulate macrophage polarization by regulating the JAK/STAT pathway to reduce ulcerative colitis and obesity-associated metabolic disorders (170, 171). MSCs also play an important role in improving ALI/ARDS by regulating macrophage polarization through STAT signaling. Xu et al. found that UC-MSCs promoted anti-inflammatory polarization and T-regulatory cell differentiation to suppress inflammatory responses and reduce LPS-stimulated lung injury (50). Meanwhile, the authors found that transfection with surfactant protein B enhanced the repair of ALI/ARDS by MSCs, which may be related to the regulation of STAT3 signaling (50).