Exosomal Non-Coding RNAs: Novel Regulators of Macrophage-Linked Intercellular Communication in Lung Cancer and Inflammatory Lung Diseases

Macrophages are innate immune cells and often classified as M1 macrophages (pro-inflammatory states) and M2 macrophages (anti-inflammatory states). Exosomes are cell-derived nanovesicles that range in diameter from 30 to 150 nm. Non-coding RNAs (ncRNAs), including microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs), are abundant in exosomes and exosomal ncRNAs influence immune responses. Exosomal ncRNAs control macrophage-linked intercellular communication via their targets or signaling pathways, which can play positive or negative roles in lung cancer and inflammatory lung disorders, including acute lung injury (ALI), asthma, and pulmonary fibrosis. In lung cancer, exosomal ncRNAs mediated intercellular communication between lung tumor cells and tumor-associated macrophages (TAMs), coordinating cancer proliferation, migration, invasion, metastasis, immune evasion, and therapy resistance. In inflammatory lung illnesses, exosomal ncRNAs mediate macrophage activation and inflammation to promote or inhibit lung damage. Furthermore, we also discussed the possible applications of exosomal ncRNA-based therapies for lung disorders.


Introduction
Resident tissue macrophages remain one of the most frequent immune cells and develop from embryonic progenitors or bone marrow monocytes [1]. The functions of resident tissue macrophages include the removal of dead cells and other cell debris, tissue immune surveillance, and maintenance of tissue homeostasis [2]. Alveolar macrophages (AMs), interstitial macrophages (IMs), and recruited macrophages are macrophages found in lung tissues. AMs shield the alveoli and airways from inhaled pathogens and particles. On the vascular and lung interstitium, IMs have immune surveillance effects [3,4]. Recruited macrophages are derived from recruited monocytes during lung inflammation [4]. Alveolar epithelial cells (AECs) serve as lung tissue barriers to protect from external and internal challenges. AMs can interact with AECs to maintain lung homeostasis [5]. Macrophages are activated in response to tissue damage and go through considerable phenotypic and functional changes to engage in tissue repair and regeneration. Additionally, dysregulated macrophage populations can impede tissue regeneration and cause pathological fibrosis [6]. Macrophages are related to various inflammatory respiratory illnesses, including acute lung injury (ALI) [7], asthma [8], and pulmonary fibrosis [9]. Currently, cancer ranks as the primary cause of disease-related deaths throughout the world [10]. The tumor microenvironment (TME), which drives the development of cancer, is complex and contains stromal cells, fibroblasts, endothelial cells, and immune cells [11]. Tumor-associated macrophages (TAMs) are macrophages that infiltrate the TME after being recruited by tumor cells. TAMs CD80, CD86, and IL-1R. M1 macrophages also release many pro-inflammatory molecules, including IL-1, IL-6, IL-12, IL-23, inducible nitric oxide synthase (iNOS), and matrix metalloproteinase 12 (MMP12) [34]. M2 macrophages have high levels of CD163, CD200R, CD204, and CD206 expression [35,36]. M2 macrophages also produce anti-inflammatory cytokines, such as IL-10, arginase-1 (Arg1), C-C motif chemokine ligand 24 (CCL24), CCL22, and others [35]. M1 macrophages are related to T helper 1 (Th1) responses, type I inflammation, bacterial killing, and tumor resistance [33]. M2a macrophages induce Th2 responses, allergy, anti-inflammatory response, and tissue remodeling [34]. M2b macrophages involve Th2 activation, tumor progression, and immunomodulation [37]. M2c macrophages lead to immunosuppression, matrix deposition, and tissue remodeling [37,38]. In summary, to accurately describe the phenotypic characterization of macrophages, it is essential to consider all described markers comprehensively. Individual markers are limited to distinguishing macrophage phenotypes, considering that distinct cellular subsets can share some molecules [39].

Exosomal ncRNAs Regulate Macrophage-Linked Intercellular Communication in Lung Cancer
Lung cancer is the most frequent cancer based on current diagnoses and the leading cause of cancer-related mortality globally [40]. Lung cancer can be divided into small cell lung cancer (SCLC) (15%) and non-small cell lung cancer (NSCLC) (85%). NSCLC contains lung adenocarcinoma (40%), lung squamous cell carcinoma (30%), and large cell carcinoma (15%) [41]. Lung cancer risk factors include genetic mutations and environmental interactions, such as cigarette smoking, radiation exposure, and toxic substances [42]. Presently, metastasis and recurrence are the main causes of death for lung cancer patients. Approximately two-thirds of patients have metastatic lesions when they are first diagnosed with lung cancer [43]. Although there are many lung cancer therapies (surgical resection, chemotherapy, radiotherapy, etc.), the overall five-year survival rate of lung cancer patients is still as low as 17.7% [44], and the overall five-year survival rate of patients with distant organ metastasis is 4.5% [45]. TAMs, together with cancer stem cells, cancer cells, fibroblasts, T cells, B cells, and natural killer (NK) cells, form an immune-suppressive TME. TAMs are related to cancer-related inflammation and drive cancer therapy tolerance, cancer recurrence, and cancer distant metastasis [46,47]. M1 TAMs inhibit lung cancer development. M2 TAMs reprogram lung TME and promote immune evasion of lung cancer cells [48]. Pritchard et al. discover that lung tumor cell-derived exosomes boost macrophage polarization into the M2 state [49]. Zhu et al. reveal that macrophage-derived apoptotic bodies strengthen lung tumor cell proliferation [50]. Additionally, exosomes derived from M2 macrophages boost drug resistance in NSCLC [51]. In summary, lung tumor cell-TAM crosstalk is a major driver of lung tumor progression. Exosomes function as essential intercellular messengers to regulate cell-to-cell communication between lung tumor cells and TAMs.

Exosomal miRNAs from Lung Tumor Cells Can Be Transferred into Macrophages
miR-19b-3p expression is higher in lung tumor cell-secreted exosomes than in lung tumor cells. miR-19b-3p targets protein tyrosine phosphatase receptor type D (PTPRD). Lung cancer cell-derived exosomal miR-19b-3p raises miR-19b-3p levels in macrophages. Then, miR-19b-3p up-regulation reduces PTPRD to promote M2 polarization of macrophages through the phosphorylation of signal transducer and activator of transcription 3 (STAT3). Chen et al. indicate that miR-19b-3p knockdown abrogates lung cancer metastasis via attenuating macrophage M2 polarization [61]. miR-181b is highly expressed in the serum of NSCLC patients. Through the activation of the Janus kinase 2 (JAK2)/STAT3 signaling pathway, exosomal miR-181b generated from A549 cells facilitates the M2 polarization of macrophages. Eventually, exosomal miR-181b potentiates lung cell proliferation, cell migration, and cell invasion [62]. miR-146a can be found in H1299 cell-released exosomes. Exosomal miR-146a from H1299 cells can be transmitted into macrophages and then elevates miR-146a expression in macrophages. Exosomal miR-146a suppresses TNF receptorassociated factor 6 (TRAF-6) and IL-1 receptor-associated kinase 1 (IRAK-1) to repress M1 macrophage polarization, which in turn enhances cell invasion and cell proliferation in H1299 cells [63]. Hypoxic TME amplifies aggressiveness and metastasis in NSCLC. The prognosis for NSCLC may be improved by targeting hypoxia [64]. Exosomes from hypoxic lung carcinoma cells contain a high concentration of miR-21. Interferon-regulatory factor 1 (IRF1) is a downstream target of miR-21. Under hypoxia, lung cancer cell exosomal miR-21 restricts IRF1 expression, which stimulates M2 macrophage polarization and ultimately contributes to lung cancer cell proliferation [65]. miR-101 is down-regulated in blood samples collected from lung cancer patients who had high levels of hypoxia-inducible factor 1α (HIF1α). miR-101 targets and inhibits cyclin-dependent kinase 8 (CDK8) [66]. The suppression of exosomal miR-101 from hypoxic A549 cells in the co-culture system raises CDK8 expression and then increases IL1A and IL6 in macrophages, resulting in macrophage inflammation. In addition, miR-101 over-expression abrogates lung cancer growth and inflammation in mice [66]. miR-770 can be packaged into exosomes secreted by A549 cells. miR-770 over-expression significantly decreases mitogen-activated protein kinase kinase kinase 1 (MAP3K1) expression in macrophages [67]. Exosomes derived by miR-770 agomir-treated A549 cells increase miR-770 expression in macrophages. miR-770 up-regulation limits macrophage M2 polarization through MAP3K1, impeding cell invasion and cell migration of A549 cells. Moreover, exosomes expressing up-regulated miR-770 also attenuate lung tumor growth in vivo [67]. Trivedi et al. suggest that dual-targeted hyaluronic acid-based nanoparticles can transfect SK-LU-1 cells with wild-type p53 (wt-p53) and miR-125b expressing plasmid DNA, thus increasing p53 and miR-125b levels in exosomes obtained from plasmid DNA-transfected SK-LU-1 cells. Exosomal wt-p53 and miR-125b reprogram M2 TAMs and lead to M2 repolarization towards the M1 state [68]. In summary, exosomal miRNAs from lung cancer cells can modify macrophage miRNA expression. In addition, disturbing the expression of lung cancer cell-derived exosomal miRNAs can reprogram macrophages. Exosomal miRNAs from lung cancer cells often promote M2 macrophage polarization, which in turn encourages lung cancer cell proliferation, cell migration, and cell invasion.

The Impacts of Exosomal circRNAs on Macrophage-Linked Intercellular Communication
Exosomal circSHKBP1 expression is up-regulated in serum from NSCLC patients. By sponging miR-1294, circSHKBP1 induces pyruvate kinase M2 isoform (PKM2) mRNA expression [83]. A549-derived exosomes expressing circSHKBP1 increase the expression of M2 polarization markers (e.g., CD206, IL-10, IL-4, and Arg1) while decreasing the levels of M1 polarization markers (i.e., CD86, IL-12, TNF-α, and INF-γ) [83]. Exosomal circSHKBP1 represses miR-1294 expression as well as enhances PKM2 expression and glucose uptake in macrophages [83]. These suggest that exosomal circSHKBP1 from A549 cells contributes to macrophage polarization from the M1 to M2 state via the miR-1294-PKM2 axis. Exosomal circSHKBP1 also accelerates lung cancer growth, metastasis, and M2 macrophage infiltration in the NSCLC mice model. Furthermore, serum exosomal circSHKBP1 might function as a biomarker for NSCLC diagnosis and prognosis [83]. Blood-derived exosomes from individuals with lung cancer contain a lot of circPVT1. Compared to normal tissues, lung cancer tissues exhibit reduced levels of miR-124-3 expression [84]. circPVT1 sponges miR-124-3p and inhibits its expression. The epigenetic regulator enhancer of zeste homolog 2 (EZH2) is a target of miR-124-3p [84]. Through the miR-124-3p-EZH2 axis, exosomal circPVT1 from A549 cells stimulates M2 macrophage polarization, which potentiates lung cancer cell proliferation, cell migration, and cell invasion. Additionally, high blood exosomal circPVT1 expression is linked to shorter survival time in lung cancer patients [84]. circFARSA is found in high concentrations in NSCLC tissues. circFARSA accelerates PTEN degradation and activates the PI3K/AKT pathway [85]. Lung tumor cell-derived exosomal circFARSA promotes M2 macrophage polarization through the PTEN/PI3K/AKT pathway, thus strengthening lung cancer cell invasion and migration [85]. Plasma circ-FARSA serves as a promising noninvasive biomarker for the diagnosis of NSCLC [86]. Following surgical resection of malignant lesions, patients with lung adenocarcinoma have decreased levels of serum exosomal circZNF451. E74-like factor 4 (ELF4) potentiates IRF transcription [87]. Exosomal circZNF451 supplied by lung tumor cells interacts with the E3 ligase TRIM56 before inducing fragile-X-related protein 1 (FXR1) ubiquitination to activate ELF4/IRF4 pathway in macrophages [87]. Thereby, exosomal circZNF451 stimulates M2 macrophage polarization to abolish the proliferation and immune functions of CD8 + T cells. Consequently, tumor exosomal circZNF451 is favorable for immune-suppressed TME and represses immunotherapy efficacy in lung cancer. Additionally, serum exosomal circZNF451 can be used as a biomarker to identify patients with lung adenocarcinoma who have a poor prognosis [87]. Compared to normal cells, the expression of circ-ADRM1 is up-regulated in lung cancer cells. Exosomal circ-ADRM1 from lung cancer cells (A549 and PC-9 cells) can enhance the M2 polarization of TAMs, which can promote lung cancer cell migration and invasion [88]. In short, exosomal circRNAs from lung cancer cells change macrophage polarized phenotypes via the miRNA-mRNA axis, which in turn supports lung cancer cell proliferation, cell migration, and cell invasion. Furthermore, circulating exosomal circRNAs function as potential noninvasive biomarkers for the diagnosis and prognosis of lung cancer patients.
Taken together, exosomal ncRNAs regulate intercellular communication between TAMs and lung tumor cells in lung TME (Figure 1), which can influence lung cancer proliferation, migration, invasion, angiogenesis, metastasis, and therapeutic resistance. Exosomal ncRNAs function as promoters or inhibitors in lung cancer (Table 1). Exosomal ncRNAs from lung tumor cells mainly induce M2 polarization of TAMs.   Tumor cells secrete exosomal ncRNAs to induce TAM polarization. TAMs deliver exosomal ncRNAs into lung cancer cells and influence cancer proliferation, migration, invasion, angiogenesis, metastasis, and therapeutic resistance.

Exosomal ncRNAs Regulate Macrophage-Linked Intercellular Communication in Acute Lung Injury
ALI is a severe respiratory syndrome with the existence of hypoxemia. Acute respiratory distress syndrome (ARDS) is a serious form of ALI. ALI and ARDS are prevalent among critically ill patients [89]. In ALI, the resident lung cells can recruit inflammatory cells (i.e., neutrophils and macrophages) into the airway microenvironment and the inflammatory responses lead to lung injury [90]. There are many risk factors associated with ALI, including direct injury (e.g., pneumonia, gastric aspiration, pulmonary contusion, alveolar hemorrhage, etc.) and indirect injury (e.g., severe sepsis, transfusions, pancreatitis, and shock) [91]. Recently, LPS-induced in vitro and in vivo models have been widely used to study ALI pathogenesis [92]. M2 macrophages attenuate lung inflammation and inhibit ALI, whereas M1 macrophages promote lung inflammation and contribute to lung tissue injury [93]. LPS-treated macrophage-derived exosomes impede the cell viability of AECs [94]. Wang et al. indicate that exosomes from LPS-treated AMs facilitate inflammatory responses and lung tissue injury in ALI mice [95].

Exosomal ncRNAs Regulate Macrophage-Linked Intercellular Communication in Pulmonary Fibrosis
Pulmonary fibrosis is a devastating lung disease featured by progressive and irreversible destruction of lung architecture, resulting in impaired gas exchange and respiratory failure [113]. Recently, mice with bleomycin (BLM) and silica treatment are often used to explore the disease progression of pulmonary fibrosis [114,115]. Current pathogenic theories proved that fibroblast to myofibroblast transition (FMT) plays a pivotal role in the pathogenesis of idiopathic pulmonary fibrosis, characterized by the abnormal generation of extracellular matrix (ECM) depositing in the lung parenchyma [116,117]. Silica-treated macrophage-derived exosomes promote fibroblast differentiation into activated myofibroblasts and augment fibroblast proliferation and migration through endoplasmic reticulum stress, which can lead to pulmonary fibrosis [118].

Exosomal ncRNAs from Other Sources Can Be Transferred into Macrophages
miR-27b-3p has been shown to induce synovial fibrosis in knee osteoarthritis [128]. Feng et al. report that repressing RGS1 promotes iNOS and IL-1β expression and elevates cathepsin and MMP activities in Flt3 + AMs [112]. Exosomal miR-27b-3p from type II AECs strengthens the pro-inflammatory and antifibrotic effects of Flt3 + AMs via decreasing RGS1. In addition, exosomes released by type II AECs increase the Flt3 + AM proportion and suppress BLM-exposed pulmonary fibrosis [112]. Recently, scientists can reprogram somatic cells to create pluripotent stem cells in a state similar to embryonic stem cells. These cells are called induced pluripotent stem cells (iPSCs) and can be used for regenerative medicine, disease modeling, and drug testing [129,130]. miR-302a-3p is up-regulated in iPSC exosomes compared to embryo fibroblast exosomes. Exosomal miR-302a-3p from iPSCs hinders M2 polarization of macrophages via targeting ten-eleven translocation 1 (TET1), resulting in repressing BLM-induced lung fibrosis [131]. Exosomal miRNAs from AECs and iPSCs can target macrophages to influence lung fibrosis.

Exosomal ncRNAs Regulate Macrophage-Linked Intercellular Communication in Asthma
Asthma is a chronic inflammatory respiratory illness affecting an estimated 334 million people in the world [132]. The symptoms of asthma include recurrent wheezing, chest tightness, cough, and shortness of breath. The main contributors to asthma pathogenesis include airway inflammation and airway remodeling [133]. Airway smooth muscle cells (ASMCs), the main structural component in the airway, affect airway inflammation via the secretion of various cytokines, chemokines, and growth factors [134]. M1 macrophages can release inflammatory factors to promote asthma. While M2 macrophages lead to inflammatory resolution and inhibit asthma [135]. Dong et al. suggest that exosomes from MSCs inhibit M1 polarization and promote M2 polarization of macrophages, thus lessening asthma in vivo [136]. miR-370 is down-regulated in lung tissues of asthmatic mice. Fibroblast growth factor 1 (FGF1) is a target of miR-370 [137]. M2 macrophage-derived exosomes containing miR-370 repress FGF1 to deactivate the MAPK/STAT1 signaling pathway, which can impede proliferation and inflammation in platelet-derived growth factor (PDGF-BB) treated ASMCs. In addition, M2 macrophage exosomal miR-370 also abrogates asthma progression in asthmatic mice [137]. Scorpion and centipede (SC) belong to insect Chinese medicine and can induce M2 macrophage polarization to relieve severe asthma [138]. M2 macrophage exosomes contain stronger miR-30b-5p expression than M1 macrophage exosomes. SC-induced M2 macrophage exosomes carrying miR-30b-5p reduce airway epithelial cell pyroptosis via restricting IRF7, resulting in restraining asthma progression [138]. EMT can be triggered by the TGF-β1/Smad3 signaling pathway and can support airway remodeling in asthma [139]. miR-21-5p is highly expressed in lung tissues of mice with ovalbumin (OVA)-induced asthma. miR-21-5p binds to Smad7 3 UTR. Exosomal miR-21-5p derived from LPS-exposed AMs activates the TGFβ1/Smad signaling pathway via downregulating Smad7, which can lead to EMT in rat tracheal epithelial cells [140]. Shang et al. reveal that mmu_circ_0001359 expression is decreased in OVA-induced asthma mice. miR-183-5p can be sponged by mmu_circ_0001359 to augment FOXO1 expression [141]. Through the miR-183-5p-FOXO1 axis, exosomal mmu_circ_0001359 from ADSCs suppresses M1 macrophage polarization and stimulates M2 macrophage polarization, resulting in dampened airway remodeling in asthma mice [141]. In summary, exosomal ncRNAs from M2 macrophages or MSCs can inhibit airway remodeling to suppress asthma progression.

Discussion and Conclusions
Macrophages are one of the most prevalent immune cells found in lung tissues. Macrophages are highly plastic cells with different phenotypes and functions, which are influenced both by their origin and resident tissue microenvironment. M1 macrophages potentiate systemic inflammatory responses and restrain tumor progression. M2 macrophages restrict inflammation and accelerate the development of tumors. The specific components of exosomes determine whether they have supportive or inhibitory functions. ncRNAs are enriched in exosomes secreted by macrophages or other cell types. Cell-to-cell communication between macrophages and target cells is governed by exosomal ncRNAs. Exosomal ncRNAs modulate macrophage polarization as well as TME reprogramming. Exosomal ncRNAs regulate macrophage-linked intercellular communication to influence lung cancer and inflammatory lung diseases. Medical applications of ncRNAs in treating human illnesses have recently gained prominence. Some miRNA-based therapies are being examined in clinical trials, such as TargomiR (miR-16 mimic-based therapy) for malignant pleural mesothelioma [142] and Miravirsen (anti-miR-122 based therapy) for hepatitis C virus infection [143]. Accumulative evidence suggests that exosomes can be processed and used as drug carriers [144,145]. Immunotherapy is currently the most promising treatment strategy against cancers since tumor immune evasion is a crucial stage in the malignant growth of tumors and one of the main obstacles to it. Macrophage-targeting therapies are emerging immune-related treatments [146]. Presently, we discovered that Chinese medicine SC (scorpion and centipede)-induced M2 macrophages can transfer exosomal miR-30b-5p into airway epithelial cells and block pyroptosis, resulting in suppressing asthma [138]. This finding points to a potential lung dysfunction therapeutic approach employing macrophage exosomal miRNAs. These days, the rational design of therapies based on the exosomal ncRNA-macrophage axis has attracted widespread attention. We hope that this review will provide new ideas for more therapies such as SC (scorpion and centipede), thereby allowing us to treat lung disorders more effectively.
Presently, despite significant advancements in the study of lung cancer and inflammatory lung illnesses, the research based on the exosomal ncRNA-macrophage axis is still lacking. Exosomal ncRNAs are critical regulators of macrophage activation, polarization, inflammation, and recruitment. In lung cancer, studies are primarily conducted using NSCLC cell lines, and studies on SCLC cell lines and in lung cancer in vivo models are still limited. In inflammatory lung disorders, it is widely suggested that LPS-induced inflammation can mimic the injurious microenvironment. Animal models induced by LPS may be useful in investigating the pathological mechanism of lung injury.