The delivery route, number of cells, and schedule of administration (e.g. single dose versus repeated doses) should be considered to optimize the therapeutic efficacy of IMRCs. In our previous study, intravenous administration of IMRCs improved the survival rate of mice in a BLM-induced model (in which diffuse lung lesions develop in a dose-dependent manner following accumulation of BLM in the lung interstitium) by inhibiting both pulmonary inflammation and fibrosis. IMRCs inhibited the production of pro-inflammatory cytokines and pro-fibrosis cytokines (such as tumor necrosis factor α and transforming growth factor β1) in lung tissue, and protected both alveolar type II cells and endothelial cells [21].
IMRCs were administered by intratracheal injection or intravenous injection into the tail vein to evaluate their anti-fibrotic effects against BLM-induced PF. IMRCs inhibited pulmonary inflammation and fibrosis in a dose-dependent manner, as shown in our previous study. However, inhibition was stronger in the group administered IMRCs intravenously after completion of BLM administration. Although the reasons for this are unclear, it is possible that BLM toxicity impaired the function of IMRCs administered during the BLM administration period in this model. Intravenous infusion, one of the principal delivery routes for therapeutics, is considered to be minimally invasive and simple-to-use, and is the most common mode for MSCs delivery in diverse lung disorders[9]. Furthermore, systemic intravenous transplantation may be a suitable administration route for future clinical scenarios.
MSCs administered during the inflammation phase of PF might be negatively affected by inflammatory cytokines once recruited to the highly inflammatory lung tissue. Thus, although higher therapeutic efficacy may be obtained by administering MSCs at an early stage of disease[10], further investigation is necessary for clinical application. Early efficacy of MSCs may be related to the immunomodulatory ability of these cells, which can reduce inflammation and preserve lung epithelium and endothelium, thereby ameliorating lung fibrosis. In contrast, cells transplanted a few days post injury had no effect on subsequent collagen deposition or prevention of fibrosis, and even elicited some aberrant actions. An alternative approach is a second dose during the developmental phase of PF. In ventilator-induced lung injury, intravenous administration of 2 × 106 bone marrow-derived MSCs (BMSCs) followed by a second dose was safe and effective for enhancement of lung repair without adverse effects [11]. However, the optimal timing for repeated cell administration needs to be established. Near-infrare fluorescence (NIRF) imaging showed that the cell membrane dark red fluorescent probe fluorescence intensity declined half dramatically by day 6 [21]. Other researchers found that BMSCs may reach their therapeutic peak and produce soluble factors to ameliorate pulmonary fibrosis 2–3 days after administration[12]. Based on these findings, a second dose of IMRCs was administered 6 days later (7 days after BLM instillation). The results showed that IMRCs (5 × 106 cells/mouse on day 1 and 2.5 × 106 cells/mouse on day 7) clearly reduced the extent of fibrosis in histological analyses. Furthermore, lung function parameters such as PV curves, IC, Rrs, Crs, Ers, Rn, G, H, and FVC were improved following IMRCs transplantation compared with those in the BLM group. These findings indicated that administration of 5 × 106 cells/mouse at day 1 and 2.5 × 106 cells/mouse at day 7 was the optimal dose and timing.
Injury to alveolar epithelial cells contributes to the pathogenesis of BLM-induced PF[13, 14]. Thus, strategies that promote the proliferation or replenishment of damaged alveolar epithelial cells may inhibit PF. Previous studies demonstrated that BMSCs adopt the morphological and molecular phenotypes of alveolar type I or II cells to repair damaged lung and reduce PF[15, 16]. However, these phenotypes were not observed in our study. As indicated by our in vivo findings, IMRCs homed to the lungs, but stayed briefly and disappeared completely after 32 days in mouse. Furthermore, only a few IMRCs adopted specific alveolar epithelial phenotypes. These findings were consistent with previous studies[17, 18]. Our results suggested that differentiation might not be the major mechanism for IMRCs-mediated tissue repair. Indeed, the concept of IMRCs engraftment and differentiation is doubtful not only for lung diseases, but also other diseases. In a previous study, high concentrations of hepatocyte growth factor, keratinocyte growth factor, and bone morphogenetic protein-7 were observed in the medium from the silica plus BMSCs group[17]. All these components play crucial roles by accelerating alveolar epithelial cell proliferation and reversing the process of lung fibrosis. Consistent with this, quite a few studies have indicated that the beneficial effects of IMRCs may be related to paracrine mechanisms. Conditioned media from IMRCs contains various soluble factors capable of exerting powerful cytoprotective, anti-inflammatory, and anti-fibrotic effects[4]. IMRCs-derived extracellular vesicles administrated via IT or IV routes were both effective for the treatment of bleomycin-induced pulmonary fibrosis[18]. In the present study, IMRCs promoted repair of the alveolar epithelium around themselves and inhibited the fibrotic process. Histological findings in the lungs, such as cellular nodules, alveolar interstitial thickening, and collagen disposition, tended to be decreased in the IMRCs group compared with those in the BLM group. Correspondingly, expression of COL-I, FN, and ⍺-SMA was downregulated. Expression of alveolar epithelial markers (HOPX and SPC) was significantly upregulated in lung tissues where IMRCs focused. In a previous study, conditioned media from BMSCs protected damaged epithelial cells and attenuated BLM-induced pulmonary fibrosis[18]. Taken together, the available in vivo and in vitro data converge to suggest that the protective effects of IMRCs are not attributable to their differentiation into lung cell phenotypes, but instead rely on paracrine mechanisms through released factors to alleviate the lung injury induced by BLM.
There is evidence that the regulation of macrophages is closely related to CD24 molecules. CD24, also known as heat stable antigen or small cell lung cancer cluster 4 antigen, is a highly glycosylated glycosylphosphatidylinositol anchored surface protein. CD24 can serve as a “don’t eat me” signal to participate in the immune escape of cancer cells. In 2019, researchers such as Barkal reported that CD24 expressed in tumors interacts with the inhibitory receptor sialic acid binding Ig-like lectin 10 (Siglec-10) of macrophages, causing rearrangement of the cytoskeleton of macrophages, thereby blocking Toll-like receptor (TLR) mediated inflammation and cellular phagocytosis, leading to immune escape[19]. CD24 can inhibit the k-light chain enhancement (NF-kB) pathway and cytokine/chemokine production of nuclear factor activated B cells. CD24 can also interact with Siglec-10, inhibiting the destructive inflammatory response of macrophages to infection [20], sepsis[21], liver injury[22], and chronic graft versus host disease. Recently, Israeli scientists carried out the Phase I clinical trial (NCT04747574) for the treatment of novel coronavirus infection by preparing CD24 positive exosomes (EXO-CD24). After treatment with EXO-CD24, the inflammatory storm was suppressed, and 29 out of 30 patients were discharged. The above indicates that CD24 plays an important role in the regulation process of macrophages.
There are several limitations in this study. First, although the mouse BLM model phenotype is similar to the acute inflammatory phase of human ARDS, observations of fibroblastic foci, alveolar epithelial type Ⅱ cells hyperplasia, and honeycombing lesions were reduced compared with those in humans, indicating that the reproduction of human IPF was not complete in our mouse BLM model. In this study, IMRCs were administered at 1 and 7 days after initiation of BLM, when fibrosis was not developed. Several previous studies reported that MSCs administration did not improve pathologically established PF. Regarding the efficacy of IMRCs during the fibrosis phase, further studies are necessary and our investigation is ongoing. In addition, some studies suggest that MSCs exacerbate pulmonary fibrosis[23]. Although it remains unclear whether MSCs promote PF, their lack of an effect may result from differences in fibrosis stage after induction, species of model animals, and administration of MSCs in the fibrosis growth phase rather than the inflammatory phase. Indeed, because the IPF-specific cellular and molecular mechanisms of MSCs have yet to be sufficiently clarified, clinical application should be considered carefully.